A new test to detect antibodies against Mycobacterium tuberculosis complex in red deer serum

The Veterinary Journal 244 (2019) 98–103

Contents lists available at ScienceDirect

The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl

A new test to detect antibodies against Mycobacterium tuberculosis complex in red deer serum J. Thomasa,b,1, J.A. Infantes-Lorenzoc,d,1, I. Morenoe, B. Romeroc, J.M. Garridof , R. Justeg , M. Domíngueze , L. Domínguezc,d, C. Gortazara,* , M.A. Risaldeh,i a

SaBio (Health and Biotechnology), Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM), Ciudad Real, Spain Indian Council of Agricultural Research (ICAR), New Delhi, India VISAVET Health Surveillance Centre, Complutense University of Madrid, Madrid, Spain d Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain e Unidad de Inmunología Microbiana, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain f NEIKER-Tecnalia, Animal Health Department, Derio, Bizkaia, Spain g Servicio Regional de Investigación y Desarrollo Agrario (SERIDA), Villaviciosa, Asturias, Spain h Dpto. de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Universidad de Córdoba (UCO), Agrifood Excellence International Campus (ceiA3), Córdoba, Spain i Infectious Diseases Unit, Instituto Maimonides de Investigación Biomédica de Córdoba (IMIBIC), Hospital Universitario Reina Sofía de Córdoba, Universidad de Córdoba, Cordoba, Spain b c

A R T I C L E I N F O

A B S T R A C T

Article history: Accepted 17 December 2018

Red deer (Cervus elaphus) farming is a growing economic activity worldwide. However, the capacity of this species to act as reservoir of animal tuberculosis (TB) poses a threat to other wildlife and to livestock. Diagnostic assay accuracy in this species is therefore highly relevant for prevention and control measures. Our aim was to evaluate the diagnostic performance of the protein complex P22, obtained from Mycobacterium bovis derived purified protein derivative (bPPD), as a candidate antigen for the detection of antibodies against Mycobacterium tuberculosis complex (MTC). We assessed the performance of this new antigen in indirect enzyme-linked immunosorbent assays (ELISA) in TB-positive and TB-negative red deer, in comparison with a bPPD-based ELISA. The P22 ELISA achieved a higher specificity (Sp) and similar sensitivity (Se) in comparison with the bPPD ELISA at all the cut-off points considered. The P22 ELISAyielded optimal Sp (99.02%; 95% confidence intervals [CI95%]: 96.5–99.8) and appropriate Se (70.1%; CI95%: 63.6–76) at the selected cut-off point of 100%. These results suggest that P22 can be used as an alternative antigen in the immunodiagnosis of animal TB through the use of an ELISA-type detection of antibodies against MTC in red deer, thus contributing to the diagnosis of animal TB in this species as a measure for further disease prevention and control programs. © 2018 Published by Elsevier Ltd.

Keywords: Bovine PPD Cervids P22 Serology Tuberculosis diagnosis

Introduction Animal tuberculosis (TB) is a multi-host disease shared by domestic and wild animals that is principally caused by Mycobacterium bovis (M. bovis) and other closely related members of the Mycobacterium tuberculosis complex (MTC). There is a wide range of wildlife reservoirs, out of which various species of deer can act as maintenance hosts. In Europe, the red deer (Cervus elaphus) is a MTC maintenance host at least in the southwestern Iberian Peninsula and the Alpine range (Gortazar et al., 2015). Red deer also play a crucial epidemiological role as long-living spillover hosts in New

* Corresponding author. E-mail address: [email protected] (C. Gortazar). 1 Both first authors contributed equally to this work. https://doi.org/10.1016/j.tvjl.2018.12.021 1090-0233/© 2018 Published by Elsevier Ltd.

Zealand (Nugent, 2011). TB is one of the main health problems affecting the deer farming industry (Mackintosh et al., 2004). One of the most important TB control measures is an accurate diagnosis (Busch et al., 2017) and subsequent culling of infected animals. The current diagnostic techniques employed to detect TB in red deer are based on assessing the cell-mediated immune response elicited against MTC, either in vivo (skin test) or in vitro (IFN-g test). The standard ante mortem screening test approved by the World Organisation for Animal Health for TB detection in farmed deer is the single intradermal tuberculin test (SITT),2 which is reported to have a sensitivity (Se) of 82–86% and a specificity (Sp) of 46–76% in deer (Palmer et al., 2011). In addition to the low 2 See: World Organization for Animal Health (WOAH-OIE), 2009: manual of diagnostic tests and vaccines for terrestrial animals. Bovine tuberculosis. http:// www.oie.int/doc/ged/D7710.PDF. (Accessed 17 December 2018).

J. Thomas et al. / The Veterinary Journal 244 (2019) 98–103

specificity, the SITT has some drawbacks, including the need to handle the animals twice in a 72-h period (Harrington et al., 2008), potential cross-reactions with environmental non-tuberculous mycobacteria (Queirós et al., 2012) and technical variability when reading the results (Fernández-de-Mera et al., 2009). The IFN-g test, which also detects the cell-mediated immune response, requires only a single blood sample, but is more demanding in terms of logistics and operator skills (Palmer et al., 2004; Waters et al., 2006; Risalde et al., 2017). As the disease progresses, the cell-mediated immune response tends to decrease at the same time as an antibody response develops (Welsh et al., 2005; McNair et al., 2007). Antibody detection techniques like enzyme-linked immunosorbent assays (ELISA) and immunochromatographic rapid tests may, therefore, be useful as alternative methods or parallel tests for cellular based assays of TB diagnosis in deer (Waters et al., 2011a; García-Bocanegra et al., 2012; Che-Amat et al., 2016). Table 1 presents the serological assays used in red deer or elk. M. bovis derived purified protein derivative (bPPD) is the most frequent antigen cocktail used in TB ELISAs (Bezos et al., 2014), but can give rise to false-positive TB results because of the sequence homology between M. bovis and other environmental mycobacteria such as Mycobacterium avium proteins (Borsuk et al., 2009; Infantes-Lorenzo et al., 2017). More specific antigens, such as MPB70, MPB83, ESAT-6 or CFP10, have been tested in serological assays in order to improve Sp (Chambers, 2009). A new immunopurified subcomplex protein from bPPD, denominated P22 (CZ Veterinaria SL, Porriño, Spain) and which is composed of several antigens including MPB70, MPB83, ESAT-6 and CFP-10, has recently proven to be a possible suitable alternative to bPPD

99

(Infantes-Lorenzo et al., 2017). This work showed that P22 provided a greater Sp in diagnostic assays when compared to bPPD, along with a similar or even greater Se for the detection of antibodies against MTC in mice. We therefore hypothesized that P22 would provide higher Sp in diagnostic tests without impacting Se levels already achieved with bPPD in red deer and other mammals. The objective of the present study was to evaluate the diagnostic performance of P22 as a candidate for use in an in-house indirect ELISA for red deer, in comparison to bPPD. Materials and methods Collection of samples We sampled the blood from 425 hunted/captured red deer (221 TB-positive and 204 TB-negative) to assess the Sp and Se in ELISA based assays. Blood samples were collected in tubes without additives from all animals using puncture of the cavernous sinus of the dura mater (Jiménez-Ruiz et al., 2016). A detailed postmortem examination was carried out in order to detect the presence of TBcompatible lesions (TBL). To perform microbiological culture, tissue samples were collected such as head lymphoid tissues, lung, tracheobronchial and mediastinal lymphoid nodes (LNs), spleen, and mesenteric LNs. Lymph nodes from additional locations or other organs were collected when any suspicious lesion was observed. Samples were stored at 20  C until analysis. Culture of samples was performed after decontamination with a final concentration of 0.37% hexadecylpyridinium chloride (Corner and Trajstman, 1988). Isolates were identified by PCR (Wilton and Cousins, 1992) and spoligotyping (Kamerbeek et al., 1997). Animals were confirmed as being TB-positive when TBL were present and MTC was cultured, and TB-negative in absence of TBL and MTC isolation by culture. Ethics statement The hunted-animals were not purposely killed and blood or tissue samples were not collected specifically for this study. Professional personnel collected blood

Table 1 Details of serodiagnostic tests in red deer and elk. Assay test

Species

N/ E

Number of animals (nSe + nSp)

Antigens

Sensitivity (Se) (%)

Specificity (Sp) (%)

References

Enzyme linked immunosorbent assay (ELISA)

Red deer

N

6 + 15

N

104 + 56

Red deer Elk

N N

94 + 217 108 + 48

Red deer Red deer and elk Elk Red deer-elk hybrid Elk

E N N E

15 + 15 16 33 + 450 10

72.7a 100 88 80 45.7 51.9 49.1 86.7 81 40 33.33

100a 100 52 79 100 100 97.9 93.3

García-Bocanegra et al. (2012)

Red deer

bPPD MPB83 bPPD MPB70 bPPD, MPB 70, aPPD MPB70 MPB83 Extract of M. bovis MPB70 MPB70 MPB70

N

34 + 141

82

93

Waters et al. (2011b)

Elk

N

33 + 450

62

87

Shury et al. (2014)

Elk

N

31 + 842

87.1

98.3

Nelson et al. (2012)

Red deer-elk hybrid Red deer

E

10 52 + 105

Mixed deer sp.

N/ E N

7 + 425

Elk

N

34 + 141

Red deer

N/ E N E

52 + 105

ESAT-6, CFP10, MPB83 ESAT-6, CFP10, MPB83 ESAT-6, CFP10, MPB83 ESAT-6, CFP10, MPB83 ESAT-6, CFP10, MPB83 ESAT-6, CFP10, MPB83 ESAT-6, CFP10, MPB83 ESAT-6, CFP10, MPB83 M. bovis antigensb M. bovis antigensb

Fluorescence polarization assay (FPA)

Cervid TB STAT-PAK lateral-flow test

Innovative dual-path platform technology

Multiantigen print immunoassay (MAPIA)

Elk Red deer-elk hybrid

34 10

81

72.5

Griffin et al. (1991) Griffin et al. (1994) Kang et al. (2016) Wadhwa et al. (2013) Surujballi et al. (2009) Shury et al. (2014) Harrington et al. (2008)

Harrington et al. (2008)

86.5

83.8

Buddle et al. (2010)

85.7a

94.8a

Gowtage-Sequeira et al. (2009)

79

98

Waters et al. (2011b)

84.6

91.4

Buddle et al. (2010)

82 76.7

Waters et al. (2011b) Harrington et al. (2008)

N/E – natural or experimental infection, nSe – number of TB-positive animals used for evaluation of Se, nSp – number of negative animals used for evaluation of Sp. a Se and Sp were evaluated based on mixed species of animals. b ESAT-6, CFP10, MPB59, MPB64, MPB70, MPB83, the 16-kDa protein, the 38-kDa protein, two fusion proteins comprising CFP10/ESAT-6 and the 16-kDa protein/MPB83, and two native antigens, bovine PPD and M. bovis culture filtrate.

100

J. Thomas et al. / The Veterinary Journal 244 (2019) 98–103

and tissue samples from hunted-harvested red deer during the hunting season. These animals were legally hunted under Spanish and EU legislation and all the hunters had hunting licenses. No ethical approval was deemed necessary; the collection of all the samples was performed for routine procedures before the design of this study in compliance with the Ethical Principles in Animal Research. Protocols, amendments and other resources were established and applied according to the guidelines approved by each autonomous government following the R.D.1201/2005 of the Spanish Ministry of Presidency. Serum antibody detection techniques Sera obtained by centrifugation (3000  g for 10 min) from blood without additives were stored at 20  C until testing for MTC antibody detection. Indirect bPPD ELISA The serum samples were tested in duplicate by means of an in-house ELISA using bPPD as an antigen and protein G horseradish peroxidase as a conjugate by applying the protocol described by Che-Amat et al. (2016). Briefly, after coating the plates with bPPD solution (CZ Veterinaria) at a concentration of 120 mg/ml in carbonate–bicarbonate buffer (Sigma–Aldrich) overnight at 4  C, the wells were washed with phosphate buffered saline (PBS) solution containing 0.05% Tween-20 (PBST) and blocked with 5% skimmed milk powder solution in PBS (SM) for 1 h at room temperature (RT). Sera were added in duplicate at a dilution of 1:10 in SM, incubated for 1 h at 37  C and subsequently washed three times with PBST. Protein G horseradish peroxidase (HRP)-conjugated (Sigma–Aldrich) was later added at a concentration of 0.002 mg/ml in PBS and the plates were incubated for 1 h at RT. After four washes with PBST, the color was developed with o-phenylenediaminedihydrochloride substrate (FAST OPD, Sigma–Aldrich) incubated for 20 min in darkness and at RT. The reaction was stopped with H2SO4 3N and the optical density (OD) was measured in a spectrophotometer at 492 nm. The anti-PPD–positive serum originated from red deer previously confirmed as M. bovis positive based on a mycobacterial culture; while the negative control was obtained from TB free red deer previously confirmed as M. bovis culture negative from TB-free areas. Positive and negative controls were tested in quadruplicate on every plate. Indirect P22 ELISA The indirect P22 ELISA detects antibodies against MTC based on the use of P22, derived from bPPD, as a coating antigen. The preparation of this new immunopurified subcomplex protein includes the hyperimmunisation of BALB/c mice with bPPD (CZ Veterinaria) to produce hybridome which secretes an antibody against MPB70 and MPB83 proteins. This monoclonal antibody was conjugated to a HiTrap NHS-activated HP column (GE Healthcare) according to the manufacturer’s protocol. The column was loaded with bPPD and the complex containing MPB70 and MPB83, which we named P22, was immunopurified (Infantes-Lorenzo et al., 2017). The ELISA protocol used for P22 is the same as the one described above for bPPD, except that the plates were coated with P22 at a concentration of 10 mg/ml. The positive and negative controls used were the same as those employed for bPPD. Data treatment The ELISA results were expressed as an ELISA percentage (E%), calculated by the formula as follows:

sample E% = (mean sample OD/2  mean of negative control OD)  100 The cut-off value was calculated using a ROC analysis and was defined as the value at which the highest sum of sensitivity plus specificity was obtained (Aurtenetxe et al., 2008). Discriminatory indices for sensitivity and specificity were also calculated as the ratio between each and the complementary of the other i.e. Se/(1-Sp) and Sp/(1-Se), respectively, in order to better assess the tests performances. The Se and Sp with 95% confidence intervals (CI95), along with the positive and negative predictive values (PPV and NPV, respectively), were calculated using the Clopper-Pearson method (Epitools, Ausvet), with the presence or absence of TBL and mycobacterial culture as a reference standard. The inter-test agreement between the evaluated assays was calculated by means of Cohen’s kappa coefficient (Epitools, Ausvet). All statistical analyses were performed at a confidence level of 95%.

Results The ROC analysis evidenced the diagnostic value of bPPD and P22 ELISA in red deer (Fig. 1), showing that the P22 ELISA yielded a slightly better sum of Se + Sp (170) than the bPPD ELISA (165). The P22 Sp discriminatory index doubled that for bPPD, while the Se discriminatory indexes were similar for both techniques. The P22

ELISA with a cut-off value of 100 E% had a higher Sp (99.02%) than the bPPD ELISA (91.67%). Both ELISAs had similar Se values at the various cut-off points evaluated. The data including Se, Sp, PPV, NPV and area under the curve (AUC), using confidence intervals of 95% (CI95%) for both ELISAs with a chosen cut-off value of 100, are summarized in Table 2. Table 3 presents Cohen’s kappa coefficient of agreement with CI95 between the evaluated assays (bPPD and P22 ELISAs, and TBL and MTC culture). Discussion The results obtained in this study confirmed our initial hypothesis, namely that P22 yields a higher Sp (99%) when compared with bPPD (91.6%), while maintaining the same Se (70.1% for both P22 and bPPD), for the serodiagnosis of TB in red deer. These results suggest that antibody detection tests are valuable tools for the diagnosis of TB in cervids for disease prevention and control by enabling the setup of test-and-slaughter programs or monitoring the effects of TB control measures in the field. The main problem associated with TB immunodiagnosis in ruminants is the lack of appropriate Sp, principally compromised by an exposure to other saprophytic and environmental mycobacteria (Buddle et al., 2010; Queirós et al., 2012), or M. avium paratuberculosis infection or vaccination (Buddle et al., 2010). Some authors have previously suggested that the effects of skin testing could also lead to the diagnosis of false positive animals to MTC antibodies by ELISA due to prior sensitization with bPPD, which can boost antibody responses to mycobacterial antigens (Buddle et al., 2010); however, repeated comparative skin testing in red deer at a six-month interval confirmed that it did not affect skin test results or serological responses measured by ELISA (Che-Amat et al., 2016). In this study, we tested the new immunopurified antigen P22 (which was derived from bPPD, as an alternative to bPPD itself) on red deer. This was done in order to detect antibodies against MTC, since it has already been reported to share fewer proteins with avian PPD (aPPD) than bPPD, which resulted in a greater Sp in mice (Infantes-Lorenzo et al., 2017) and pigs (Cardoso-Toset et al., 2017). In red deer, the P22 ELISA achieved a higher Sp in comparison with bPPD ELISA at all the cut-off points evaluated. Furthermore, this Sp for P22 ELISA was found to be higher than for most of the serodiagnostic tests reported previously in deer (81–98%; see Table 1). Thus, the new test allowed reducing immune cross-reactions and the diagnosis of false negative animals. However, the specificity of serological tests may be underestimated given that some tissues negative by culture may actually be infected, especially in absence of lesions. In addition, the decontamination step prior to MTC culture can adversely affect the viability of mycobacteria, especially when the number of viable microorganisms is low, and it may, therefore, lead to false negative results in the culture, which could affect the true Sp of the test (Stewart et al., 2013). Both ELISAs substantially agreed with the TBL and MTC culture; however, no antibody response was recorded in around 30% of the animals with TBL and MTC isolation in both ELISAs. In this respect, the Se of the diagnosis is very variable, which could be related to the reference technique used to classify the TB status of the animals, the animal’s immune response and its influence on antigen recognition (Marassi et al., 2011) and, most importantly, to the stage of the disease (Griffin et al., 1991; Welsh et al., 2005; McNair et al., 2007). In our study, the P22 ELISA achieved a Se as high as the bPPD ELISA and, in addition, the kappa values obtained for both tests also indicated a good agreement. This coincides with results obtained in previous research, reporting that out of 10 most abundant proteins identified in bPPD and P22, seven proteins are common,

J. Thomas et al. / The Veterinary Journal 244 (2019) 98–103

101

Fig. 1. Diagnostic value graphics for the tuberculosis serodiagnosis of ELISA in red deer when using the bovine purified protein derivative (bPPD) or the immunopurified P22 as an antigen. Sensitivity (Se), specificity (Sp) and their semi-sum are the percentages on the primary Y-axis. Discriminatory indexes for Se [Se/(1-Sp)] and Sp [Sp/(1-Se)] are on the secondary Y-axis.

Table 2 Sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV) and area under the curve (AUC) with 95% confidence intervals (CI95) in the chosen cut-off value of 100 for bPPD and P22 ELISA in red deer. Test

Se %

Cl95

%

Cl95

%

Cl95

%

Cl95

bPPD ELISA P22 ELISA

70.1 70.1

63.6–76 63.6–76

91.6 99

86.9–95 96.5–99.8

90.1 98.7

84.6–94.1 95.4–99.8

73.9 75.3

68–79.2 69.7–80.4

Sp

PPV

NPV

AUC Cl95 0.88 0.89

0.8–0.9 0.8–0.9

Table 3 Cohen’s kappa coefficient (K) with 95% confidence intervals (CI95) of agreement between the evaluated assays for TB diagnosis in red deer. Test

bPPD ELISA Kappa value

bPPD ELISA P22 ELISA

1 0.62

P22 ELISA Cl95 0.52–0.72

Kappa value 1

TBL and MTC isolation Cl95

Kappa value

Cl95

0.61 0.68

0.53–0.68 0.61–0.74

Poor agreement: K < 0.20; fair agreement: K = 0.21–0.4; moderate agreement: K = 0.41–0.6; good agreement: K = 0.61–0.8; very good agreement: K = 0.81–1. TB: tuberculosis; TBL: tuberculosis compatible lesions; MTC: Mycobacterium tuberculosis complex.

102

J. Thomas et al. / The Veterinary Journal 244 (2019) 98–103

but differing in their relative abundance and five of those proteins are predicted to be highly immunogenic (Infantes-Lorenzo et al., 2017). This implies that the vast majority of antibodies induced by MTC infection should be detected by using a serodiagnostic test based on bPPD or P22, as evidenced in mice (Infantes-Lorenzo et al., 2017) and cattle (Casal et al., 2017). In this context, the P22/bPPD ELISA could be interpreted in parallel to traditional CMI-based techniques, in order to improve the Se, as reported in cattle (Casal et al., 2017). Moreover, ELISA technique is considered a relatively low-cost diagnostic tool that is easy to perform; likewise, by allowing a large number of samples to be processed in a short time, it is routinely used in epidemiological surveillance of domestic and wild species (Boadella et al., 2011). Conclusion The new protein complex, immunopurified from bPPD, called P22, is a better candidate antigen in an indirect ELISA for the diagnosis of TB in red deer than bPPD. It provides a greater Sp and a similar Se. Therefore, we propose the use of P22 as an alternative antigen in TB immunodiagnosis through the use of an ELISA-type detection of antibodies against MTC in red deer. This will enable assessing the TB status of farmed or wild red deer as a measure for further disease prevention and control programs. Conflict of interest None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. Acknowledgements Preliminary results were presented as a Poster at XXII Simposio, Avedila, Valladolid, 16–17 November 2017. Research funding was provided by ‘Plan Nacional’ grant AGL2014-56305 (MINECO, Spain and FEDER). J. Thomas was supported by a grant from the Indian Council of Agricultural Research-International Fellowship 2014-15 (ICAR-IF 2014-15). J.A. Infantes was supported by a FPU contractfellowship (FPU2013/6000) (Ministry of Education, Culture and Sport, Spain). M.A. Risalde holds a ‘Juan de la Cierva program’ contract (IJCI-2014-19961) (Ministry of Economy and Competitiveness, Spain). The authors would like to thank D. GonzálezBarrio and F. Talavera and other IREC staff for their technical assistance. We are also thankful to Medianilla Red Deer Genetics and Sabiotec for providing valuable samples. References Aurtenetxe, O., Barral, M., Vicente, J., de la Fuente, J., Gortázar, C., Juste, R.A., 2008. Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovis in European wild boar. BMC Vet. Res. 4, 43. Bezos, J., Casal, C., Romero, B., Schroeder, B., Hardegger, R., Raeber, A.J., López, L., Rueda, P., Domínguez, L., 2014. Current ante-mortem techniques for diagnosis of bovine tuberculosis. Res. Vet. Sci. 97, 44–52. Boadella, M., Lyashchenko, K., Greenwald, R., Esfandiari, J., Jaroso, R., Carta, T., Garrido, J.M., Vicente, J., de la Fuente, J., Gortázar, C., 2011. Serologic test for detecting antibodies against Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis in Eurasian wild boar (Sus scrofa scrofa). J. Vet. Diagn. Invest. 23, 77–83. Borsuk, S., Newcombe, J., Mendum, T.A., Dellagostin, O.A., McFadden, J., 2009. Identification of proteins from tuberculin purified protein derivative (PPD) by LC–MS/MS. Tuberculosis 89, 423–430. Buddle, B.M., Wilson, T., Denis, M., Greenwald, R., Esfandiari, J., Lyashchenko, K.P., Liggett, S., Mackintosh, C.G., 2010. Sensitivity, specificity, and confounding factors of novel serological tests used for the rapid diagnosis of bovine tuberculosis in farmed red deer (Cervus elaphus). Clin. Vaccine Immunol. 17, 626–630. Busch, F., Bannerman, F., Liggett, S., Griffin, F., Clarke, J., Lyashchenko, K.P., Rhodes, S., 2017. Control of bovine tuberculosis in a farmed red deer herd in England. Vet. Rec. 180, 68.

Cardoso-Toset, F., Luque, I., Carrasco, L., Jurado-Martos, F., Risalde, M.Á., Venteo, Á., Infantes-Lorenzo, J.A., Bezos, J., Rueda, P., Tapia, I., et al., 2017. Evaluation of five serologic assays for bovine tuberculosis surveillance in domestic free-range pigs from southern Spain. Prev. Vet. Med. 137, 101–104. Casal, C., Infantes, J.A., Risalde, M.A., Díez-Guerrier, A., Domínguez, M., Moreno, I., Romero, B., de Juan, L., Sáez, J.L., Juste, R., et al., 2017. Antibody detection tests improve the sensitivity of tuberculosis diagnosis in cattle. Res. Vet. Sci. 112, 214– 221. Chambers, M.A., 2009. Review of the diagnosis and study of tuberculosis in nonbovine wildlife species using immunological methods. Transbound. Emerg. Dis. 56, 215–227. Che-Amat, A., Risalde, M.Á., González-Barrio, D., Ortíz, J.A., Gortázar, C., 2016. Effects of repeated comparative intradermal tuberculin testing on test results: a longitudinal study in TB-free red deer. BMC Vet. Res. 12, 184. Corner, L.A., Trajstman, A.C., 1988. An evaluation of 1-hexadecylpyridinium chloride as a decontaminant in the primary isolation of Mycobacterium bovis from bovine lesions. Vet. Microbiol. 8, 127–134. Fernández-de-Mera, I.G., Vicente, J., Höfle, U., Fons, F.R., Ortiz, J.A., Gortazar, C., 2009. Factors affecting red deer skin test responsiveness to bovine and avian tuberculin and to phytohaemagglutinin. Prev. Vet. Med. 90, 119–126. García-Bocanegra, I., de Val, B.P., Arenas-Montes, A., Paniagua, J., Boadella, M., Gortázar, C., Arenas, A., 2012. Seroprevalence and risk factors associated to Mycobacterium bovis in wild artiodactyl species from southern Spain, 2006– 2010. PLoS One 7, e34908. Gortazar, C., Che-Amat, A., O’Brien, D., 2015. Open questions and recent advances in the control of a multi-host infectious disease: animal tuberculosis. Mammal Rev. 45, 160–175. Gowtage-Sequeira, S., Paterson, A., Lyashchenko, K.P., Lesellier, S., Chambers, M.A., 2009. Evaluation of the CervidTB STAT-PAK for the detection of Mycobacterium bovis infection in wild deer in Great Britain. Clin. Vaccine Immunol. 16, 1449– 1452. Griffin, J.F., Nagai, S., Buchan, G.S., 1991. Tuberculosis in domesticated red deer: comparison of purified protein derivative and the specific protein MPB70 for in vitro diagnosis. Res. Vet. Sci. 50, 279–285. Griffin, J.F.T., Cross, J.P., Chinn, D.N., Rodgers, C.R., Buchan, G.S., 1994. Diagnosis of tuberculosis due to Mycobacterium bovis in New Zealand red deer (Cervus elaphus) using a composite blood test and antibody assays. N. Z. Vet. J. 42, 173– 179. Harrington, N.P., Surujballi, O.P., Prescott, J.F., Duncan, J.R., Waters, W.R., Lyashchenko, K., Greenwald, R., 2008. Antibody responses of cervids (Cervus elaphus) following experimental Mycobacterium bovis infection and the implications for immunodiagnosis. Clin. Vaccine Immunol. 15, 1650–1658. Infantes-Lorenzo, J.A., Moreno, I., Risalde, M.A., Roy, A., Villar, M., Romero, B., Ibarrola, N., de la Fuente, J., Puentes, E., de Juan, L., et al., 2017. Proteomic characterization of bovine and avian purified protein derivatives and identification of specific antigens for serodiagnosis of bovine tuberculosis. Clin. Proteomics 14, 36. Jiménez-Ruiz, S., Arenas-Montes, A., Cano-Terriza, D., Paniagua, J., Pujols, J., Miró, F., Fernández-Aguilar, X., González, M.Á., Franco, J.J., García-Bocanegra, I., 2016. Blood extraction method by endocranial venous sinuses puncture in hunted wild ruminants. Eur. J. Wildl. Res. 62, 775–780. Kamerbeek, J., Schouls, L.E.O., Kolk, A., Van Agterveld, M., Van Soolingen, D., Kuijper, S., Bunschoten, A., Molhuizen, H., Shaw, R., Goyal, M., et al., 1997. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35, 907–914. Kang, S.S., Byeon, H.S., Ku, B.K., Kim, S.W., Kim, J., Woo, J., Ahn, B., Kim, S., Monoldorova, S., Park, C.H., et al., 2016. Seroprevalence of tuberculosis in domesticated elk (Cervus canadensis) in Korea. Res. Vet. Sci. 107, 228–232. Mackintosh, C.G., De Lisle, G.W., Collins, D.M., Griffin, J.F.T., 2004. Mycobacterial diseases of deer. N. Z. Vet. J. 52, 163–174. Marassi, C.D., Medeiros, L., McNair, J., Lilenbaum, W., 2011. Use of recombinant proteins MPB70 or MPB83 as capture antigens in ELISAs to confirm bovine tuberculosis infections in Brazil. Acta Trop. 118, 101–104. McNair, J., Welsh, M.D., Pollock, J.M., 2007. The immunology of bovine tuberculosis and progression toward improved disease control strategies. Vaccine 25, 5504– 5511. Nelson, J.T., Orloski, K.A., Lloyd, A.L., Camacho, M., Schoenbaum, M.A., RobbeAusterman, S., Thomsen, B.V., Hall, S.M., 2012. Evaluation of serodiagnostic assays for Mycobacterium bovis infection in elk, white-tailed deer, and reindeer in the United States. Vet. Med. Int. doi:http://dx.doi.org/10.1155/2012/563293. Nugent, G., 2011. Maintenance, spillover and spillback transmission of bovine tuberculosis in multi-host wildlife complexes: a New Zealand case study. Vet. Microbiol. 151, 34–42. Palmer, M.V., Waters, W.R., Whipple, D.L., Slaughter, R.E., Jones, S.L., 2004. Analysis of interferon-g production by Mycobacterium bovis infected white-tailed deer (Odocoileus virginianus) using an in-vitro based assay. J. Vet. Diagn. Invest. 16, 16–20. Palmer, M.V., Whipple, D.L., Payeur, J.B., Bolin, C.A., 2011. Use of the intradermal tuberculin test in a herd of captive elk (Cervus elaphus nelsoni) naturally infected with Mycobacterium bovis. J. Vet. Diagn. Invest. 23, 363–366. Queirós, J., Alvarez, J., Carta, T., Mateos, A., Ortiz, J.A., Fernandez-de-Mera, I.G., Martin-Hernando, M.P., Gortázar, C., 2012. Unexpected high responses to tuberculin skin-test in farmed red deer: implications for tuberculosis control. Prev. Vet. Med. 104, 327–334. Risalde, M.A., Thomas, J., Sevilla, I., Serrano, M., Ortiz, J.A., Garrido, M., Domínguez, L., Domínguez, C., Gortázar, C., Ruíz-Fons, F.J., 2017. Development and evaluation

J. Thomas et al. / The Veterinary Journal 244 (2019) 98–103 of an interferon gamma assay for the diagnosis of tuberculosis in red deer experimentally infected with Mycobacterium bovis. BMC Vet. Res. 13, 341. Shury, T.K., Bergeson, D., Surujballi, O., Lyashchenko, K.P., Greenwald, R., 2014. Field evaluation of three blood-based assays for elk (Cervus canadensis) naturally infected with Mycobacterium bovis. Prev. Vet. Med. 115, 109–121. Stewart, L.D., McNair, J., McCallan, L., Gordon Grant, I.R., 2013. Improved detection of Mycobacterium bovis infection in bovine lymph node tissue using immunomagnetic separation (IMS)-based methods. PLoS One 8, e58374. Surujballi, O.M., Lutze-Wallace, C., Turcotte, C., Savic, M., Stevenson, D., Romanowska, A., Monagle, W., Berlie-Surujballi, G., Tangorra, E., 2009. Sensitive diagnosis of bovine tuberculosis in a farmed cervid herd with use of an MPB70 protein fluorescence polarization assay. Can. J. Vet. Res. 73, 161. Wadhwa, A., Johnson, R.E., Mackintosh, C.G., Griffin, J.F.T., Waters, W.R., Bannantine, J.P., Eda, S., 2013. Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer (Cervus elaphus) with bovine tuberculosis. BMC Vet. Res. 9, 256. Waters, W.R., Stevens, G.E., Schoenbaum, M.A., Orloski, K.A., Robbe-Austerman, S., Harris, N.B., Hall, S.M., Thomsen, B.V., Wilson, A.J., Brannian, R.E., et al., 2011a.

103

Bovine tuberculosis in a Nebraska herd of farmed elk and fallow deer: a failure of the tuberculin skin test and opportunities for serodiagnosis. Vet. Med. Int. doi:http://dx.doi.org/10.4061/2011/953985. Waters, W.R., Buddle, B.M., Vordermeier, H.M., Gormley, E., Palmer, M.V., Thacker, T. C., Bannantine, J.P., Stabel, J.R., Linscott, R., Martel, E., et al., 2011b. Development and evaluation of an enzyme-linked immunosorbent assay for use in the detection of bovine tuberculosis in cattle. Clin. Vaccine Immunol. 18, 1882– 1888. Waters, W.R., Palmer, M.V., Slaughter, R.E., Jones, S.L., Pitzer, J.E., Minion, F.C., 2006. Diagnostic implications of antigen-induced gamma interferon production by blood leukocytes from Mycobacterium bovis-infected reindeer (Rangifer tarandus). Clin. Vaccine Immunol. 13, 37–44. Welsh, M.D., Cunningham, R.T., Corbett, D.M., Girvin, R.M., McNair, J., Skuce, R.A., Bryson, D.G., Pollock, J.M., 2005. Influence of pathological progression on the balance between cellular and humoral immune responses in bovine tuberculosis. Immunology 114, 101–111. Wilton, S., Cousins, D., 1992. Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. Genome Res. 1, 69–273.