ORIGINAL RESEARCH published: 03 July 2020 doi: 10.3389/fvets.2020.00374 Evaluation of P22 Antigenic Complex for the Immuno-Diagnosis of Tuberculosis in BCG Vaccinated and Unvaccinated Goats Claudia Arrieta-Villegas 1*, José Antonio Infantes-Lorenzo 2 , Javier Bezos 3,4 , Miriam Grasa 5 , Enric Vidal 1 , Irene Mercader 6 , Mahavir Singh 7 , Mariano Domingo 1,8 , Lucía de Juan 3,4 and Bernat Pérez de Val 1 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Servicio de Inmunología Microbiana, Centro Nacional de Microbiología, Instituto de Investigación Carlos III, Madrid, Spain, 3 VISAVET Health Surveillance Center, Universidad Complutense de Madrid, Madrid, Spain, 4 Departamento de Sanidad Animal, Universidad Complutense de Madrid, Madrid, Spain, 5 Agrupació de Defensa Sanitària de Cabrum i Oví Lleter de Catalunya, Barbens, Spain, 6 Departament d’Agricultura, Ramaderia, Pesca i Alimentació de la Generalitat de Catalunya, Barcelona, Spain, 7 Lionex Diagnostics and Therapeutics GmbH, Braunschweig, Germany, 8 Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain Current eradication strategies of tuberculosis (TB) in goats mainly rely on the single intradermal tuberculin test (SIT) and single intradermal cervical comparative tuberculin tests (SICCTs). TB vaccination has been proposed as a cost-effective option in Edited by: Satoshi Sekiguchi, high-prevalence herds or countries where economic compensation for the slaughter of University of Miyazaki, Japan positive animals is not affordable. However, TB vaccination compromises the efficiency Reviewed by: of tuberculin-based diagnostic tests. In this study, the performance of a new diagnostic Christian Menge, platform, based on the P22 antigenic complex, was assessed for skin test (ST), Friedrich Loeffler Institute, Germany Faten Abdelaal Okda, interferon-gamma release assay (IGRA), and serology under different TB scenarios. The St. Jude Children’s Research Hospital, sensitivity (Se) of diagnostic tests was assessed in TB-infected goats from the same United States farm (herd A, N = 77). The specificity (Sp) was assessed in two TB-negative farms *Correspondence: Claudia Arrieta-Villegas (both vaccinated against paratuberculosis): one TB unvaccinated (herd B, N = 77) and claudia.arrieta@irta.cat another vaccinated with bacille Calmette-Guérin (BCG) (herd C, N = 68). The single (s) P22-IGRA showed the highest Se among IGRA tests (91%), and the comparative Specialty section: This article was submitted to (c) P22-ST showed the highest Sp (100% in herd B and 98% in herd C). Combined Veterinary Infectious Diseases, interpretation of techniques enabled the best diagnostic performances. Combining the a section of the journal SICCT + sP22-IGRA improved Se (97%) compared to SICCT + tuberculin-based IGRA Frontiers in Veterinary Science (95%), with a reduction of Sp (95 and 100%, respectively). Besides, combination of Received: 06 March 2020 Accepted: 28 May 2020 P22-ELISA with cP22-ST or SICCT elicited a similar performance in the non-vaccination Published: 03 July 2020 context (Se: 94 and 95%; Sp: 95 and 95%, respectively), but Sp was significantly higher Citation: for the combination with cP22-ST compared to SICCT in the TB vaccination context Arrieta-Villegas C, Infantes-Lorenzo JA, Bezos J, (95 and 79%, respectively). The combination of serological tests based on P22 and Grasa M, Vidal E, Mercader I, MPB83 showed higher complementarity and improved 13 percentage points the Se of Singh M, Domingo M, de Juan L and P22-ELISA alone. These findings suggest that either cell-mediated or antibody-based Pérez de Val B (2020) Evaluation of P22 Antigenic Complex for the diagnostic techniques, using the P22 antigen complex, can contribute to improve the Immuno-Diagnosis of Tuberculosis in immunodiagnostics of TB in goats under different TB control strategies. BCG Vaccinated and Unvaccinated Goats. Front. Vet. Sci. 7:374. Keywords: tuberculosis, diagnosis, goats, bacille Calmette-Guérin (BCG), skin test, interferon-gamma release doi: 10.3389/fvets.2020.00374 assay (IGRA), serology, P22 Frontiers in Veterinary Science | www.frontiersin.org 1 July 2020 | Volume 7 | Article 374 Arrieta-Villegas et al. P22 Antigen for Diagnosis of TB in Goats INTRODUCTION MATERIALS AND METHODS Tuberculosis (TB) in goats is a chronic infectious disease, mainly Herds and Experimental Design caused by Mycobacterium bovis and Mycobacterium caprae, A total of 222 goats from three herds were included in the members of the Mycobacterium tuberculosis complex (MTBC). study (Table 1): 77 infected goats (infection was confirmed This disease entails important economic costs for livestock postmortem by gross lesions, histopathology or mycobacterial industries (1) and could be a source of TB for cattle (2), other culture, or both) from a TB-positive herd of murciana-granadina domestic animals (3, 4), wildlife (5), and humans (6). goats (herd A); 77 goats belonging to an officially TB-free herd Spain has the second-highest goat census of the EU, with of alpine goats (herd B) that were vaccinated against PTB with 2.7 million goat heads (data extracted from FAOSTAT on Gudair (CZ Vaccines, Porriño, Spain), around 2 years before 17/02/2020). Besides, the high TB burden in goats could explain sampling; and 68 goats from another TB-free herd (herd C) of a number of new bovine TB breakdowns, hampering the goal Blanca de Rasquera autochthonous breed, that were vaccinated of TB eradication in cattle (7). Therefore, some regions with a against PTB (Gudair R ) and against TB with M. bovis BCG high concentration of caprine herds carry out TB eradication Danish 1,331 strain (ATCC, Ref. 35733) as described previously campaigns in caprine flocks (8); however, goat herds are still (15). In herd C, 50% of goats were vaccinated with BCG and not subjected to a national eradication program, except for those Gudair R 9–10 months before sampling, and the remaining 50% epidemiologically linked with cattle (9). were vaccinated more than 1 year before. STs, IGRAs, and The cornerstone of an efficient caprine TB eradication immunoglobulin G (IgG) enzyme-linked immunosorbent assays program is the diagnosis. The Spanish bovine TB eradication (ELISAs) were carried out in the 77 infected goats, as well as in program effectiveness is highly dependent on the routine 138, 142, and 142 noninfected goats, respectively (Table 1). tuberculin skin testing (10). Current bovine TB testing is based Two TB control scenarios were hypothesized in order to study on the single intradermal tuberculin test (SIT) and single the performance of each diagnostic test: the conventional (TB intradermal cervical comparative tuberculin tests (SICCTs), and unvaccinated) scenario, using data from herds A and B, and the the interferon-gamma release assay (IGRA). However, in goats BCG-vaccinated (TB-VAC) scenario, using data from herds A under certain epidemiological contexts, those diagnostic tests and C. Se was calculated using data from herd A, and Sp was have some drawbacks in terms of sensitivity (Se) and specificity calculated using data from herds B and C depending on TB (Sp) (8, 11). control scenario (Table 2). Another concern for TB diagnostics is the vaccination against Mycobacterium avium subsp. paratuberculosis (MAP), which Antigens has been largely implemented in small ruminants, to prevent M. tuberculosis var. bovis (PPD-B) and M. avium (PPD-A) the development of clinical disease (12). Nevertheless, even tuberculins (2,500 IU/ml) were obtained from CZ Vaccines though MAP vaccines are authorized (e.g., Gudair R vaccine), it has been demonstrated that paratuberculosis (PTB) vaccination TABLE 1 | Herd and treatment distribution of tested animals. interferes with STs and IGRA used for TB diagnosis (13, 14). In addition, the efficacy of M. bovis bacille Calmette-Guérin (BCG) No. of animals tested vaccine has also been assessed in goats during the last decade Herd TB status BCG1 Gudair®2 ST IGRA ELISA in different vaccination trials (15–19). Even though these trials showed that BCG conferred certain protection to experimentally A Positive No No 77 77 77 and naturally infected goats, it was evidenced that vaccination B Free No Yes 77 74 74 interfered with current TB diagnostic tests (16, 20). C Free Yes Yes 61 68 68 To overcome diagnostic interferences due to BCG 1 BCG, bacilli Calmette-Guérin Mycobacterium bovis vaccine. 2 Gudair vaccine, vaccine vaccination, defined antigens to differentiate infected from against paratuberculosis (Mycobacteium avium subspecies paratuberculosis). vaccinated animals (DIVA) have been developed (14, 21); TB, tuberculosis; ST, skin test; IGRA, interferon-gamma release assay. nevertheless, those antigens have shown lower Se compared to tests based on standard tuberculins (22). Recently, a new multi- protein complex called P22, obtained from purified protein TABLE 2 | TB control scenarios distribution of tested animals. derivative of M. bovis (PPD-B) by affinity chromatography, has No. of animals tested been developed (23), yielding high Se and variable Sp, depending on the animal species and epidemiological contexts (24). To Control scenario Herds BCG1 Gudair®2 ST IGRA ELISA date, this antigen has been tested to detect humoral response Conventionala A+B No Yes 154 151 151 against MTBC in different species (25–30); however, there is a TB-VACb A+C Yes Yes 138 145 145 lack of information regarding its performance for cell-mediated immunity (CMI)-based diagnostics. a Conventional scenario: composed by TB unvaccinated goats. b TB-VAC Scenario: TB The aim of this study was to evaluate the performance of negative animals from herd C were vaccinated with BCG and TB-positive animals from herd A were not vaccinated. 1 BCG, bacilli Calmette-Guérin Mycobacterium different cell-mediated and humoral immunodiagnostic tests, bovis vaccine. 2 Gudair vaccine, vaccine against paratuberculosis (Mycobacteium avium based on the P22 antigenic complex, for the diagnosis of TB in subspecies paratuberculosis). goats under different epidemiological and control scenarios. TB, tuberculosis; ST, skin test; IGRA, interferon-gamma release assay. Frontiers in Veterinary Science | www.frontiersin.org 2 July 2020 | Volume 7 | Article 374 Arrieta-Villegas et al. P22 Antigen for Diagnosis of TB in Goats and used at concentrations recommended by the Spanish 100. A sample E% <100% was classified as negative, and a sample Ministry (9). The protein complex P22 was produced by E% ≥100% was classified as positive. immunopurification of PPD-B (CZ Vaccines) as described previously (23) and prepared at a concentration of 500 µg/ml Post-mortem Examination (unpublished data). The DIVA reagent based on a cocktail of Seventy-seven goats from the positive herd (herd A) were recombinant ESAT-6 and CFP-10 proteins (500 µg/ml) (31) and euthanized after ST reading by intravenous injection of a sodium the recombinant MPB83 (MPT83) protein (500 µg/ml) (32) were pentobarbital overdose. A complete necropsy procedure was purchased from Lionex (Braunschweig, Germany). conducted for TB lesion examination. Lesions were collected and immediately fixed in 10% buffered formalin for histopathological Skin Tests confirmation by hematoxylin/eosin staining. Mediastinal and SIT was performed by intradermal inoculation of 0.1 ml of PPD- tracheobronchial lymph nodes (LNs) were removed and stored B in the left-hand side of the neck by using a Dermojet R syringe at −20◦ C for bacterial culture. (Akra Dermojet, Pau, France). In the same way, SICCT was performed by intradermal inoculation of 0.1 ml of PPD-B and Bacteriology PPD-A, both in the left-hand side of the neck, at the proximal Whole pulmonary LNs of each animal were thawed, pooled, and distal parts of the neck, respectively. Besides, 0.1 ml of P22 homogenized, and decontaminated as previously described (34) (at 500 µg/ml) was inoculated in the right-hand side of the and plated on Middlebrook 7H11 medium (BD diagnostics, neck. The increase in skinfold thickness (SFT) was measured Sparks, MD, USA). Then, cultured plates were incubated at just before the inoculation and after 72 h. Severe interpretations 37◦ C for 28 days. Finally, plates were read, and colonies were of SIT and SICCT were performed, as previously described in confirmed as MTBC by multiplex PCR (35). the manual of the Spanish bovine TB eradication program (9). Briefly, positive criterion for SIT: SFT PPD-B > 2 mm (severe); and for SICCT: positive to SIT and SFT PPD-B - SFT PPD- Data Analysis The Sp was calculated in TB-free farms (herds B and C) using the A > 1 mm (severe) or presence of clinical signs in the PPD-B formula Sp = True negatives/(True negatives + False positives). inoculation site. P22 single and comparative STs (sP22-ST and The Se was calculated in the TB-infected farm by the formula cP22-ST) were interpreted using the same criteria as SIT and Se = True positive/(True positive + False negative). Clooper- SICCT, respectively, i.e., considering SFT P22 and SFT P22 - SFT Pearson 95% confidence intervals were calculated for Sp and Se. PPD-A measures, respectively. Differences in diagnostic results, between tests, were evaluated Whole-Blood Interferon-Gamma Release by the McNemar test. Moreover, agreement between tests was calculated by Cohen’s Kappa coefficient (k) and interpreted as Assays follows: <0.00 poor, 0.00–0.20 slight, 0.21–0.4 fair, 0.41–0.60 Blood samples were collected from the jugular vein prior to moderate, 0.61–0.80 substantial, and 0.81–1.00 almost perfect. ST performance using heparinized tubes and were processed as The diagnostic performance of each test was calculated using the described previously (16). Shortly, blood samples were stimulated diagnostic odds ratio (DOR) (36). All statistical tests and 95% with PPD-B, PPD-A, and P22 at a final concentration of confidence intervals were calculated using the Epitools calculator 20 µg/ml, and with DIVA reagent (ESAT-6/CFP-10) at 20 µg/ml, (Sargento, ESG, 2018, Epitools Epidemiological Calculators, while PBS was added as an unstimulated control. Samples were Ausvet, Pty., Ltd., Australia; available in www.epitools.ausvet. incubated at 37 ± 1◦ C with 0.5% CO2 overnight. Finally, plasma com.au). supernatant was collected and analyzed by ELISA (BOVIGAM R , Thermo Fisher Scientific, Waltham, MA, USA) and read at 450 nm using a spectrophotometer (Biotek Power Wave XS). The RESULTS interpretation of tuberculin-based IGRA (STAND-IGRA) results was performed according to the cutoff point recommended by The results of Se of herd A and Sp of herds B and C are the manufacturer, i.e., the criterion for positivity: PPD-B OD – summarized in Table 3. The TB-positive status of all animals PBS OD ≥ 0.05 and PPD-B OD > PPD-A OD. Similarly, cP22- from herd A was confirmed by positive mycobacterial culture IGRA was considered positive when P22 OD – PBS OD ≥ 0.05 and/or positive lesions in histopathological analysis. and P22 OD > PPD-A OD, whereas sP22-IGRA and DIVA-IGRA were considered positive when P22 OD – PBS OD ≥ 0.05 and Skin Tests DIVA OD – PBS OD ≥ 0.05, respectively. The Se of the cP22-ST was the lowest among tests, but the Sp in herd B was the highest, being identical to the Sp of the SICCT, and Antibody Detection Tests a 6 percentage point (p.p.) and 8 p.p. more specific than the SIT Plasma samples were analyzed for antibody detection by using and the sP22-ST, respectively. Regarding the herd C, the cP22-ST two in-house indirect ELISA, one for detecting MPB83 antigen, and the sP22-ST displayed similar Sp, being significantly more performed and interpreted as described previously (33), and specific than the SIT (31 p.p. of increase, p < 0.001, and 30 p.p. another one for detecting P22, performed as described previously of increase, p = 0.005, for cP22-ST and sP22-ST, respectively) and (24, 25). P22-ELISA was interpreted as follows: ELISA percentage the SICCT (18 p.p. of increase, p = 0.0026, and 17 p.p. of increase, (E%) = [mean sample OD/(2 × mean negative control OD)] × p = 0.0094, for cP22-ST and sP22-ST, respectively). Frontiers in Veterinary Science | www.frontiersin.org 3 July 2020 | Volume 7 | Article 374 Arrieta-Villegas et al. P22 Antigen for Diagnosis of TB in Goats TABLE 3 | Sensitivity (Se) and specificity (Sp) of diagnostic tests. TABLE 4 | Sensitivity (Se) and specificity (Sp) combined results of P22-based diagnostic tests. Diagnostic TB positive Unvaccinated BCG vaccinated test (farm A) (farm B) (farm C) Diagnostic TB positive Unvaccinated BCG vaccinated tests (farm A) (farm B) (farm C) 9 N Se N Sp N Sp (95% CI)10 (95% CI11 (95% CI)11 N8 Se N Sp N Sp (95% CI)9 (95% CI)10 (95% CI)10 sP22-ST1 77 87% (77–94) 77 92% (84–97) 61 97% (89–100) cP22-ST2 77 74% (63–83) 77 100% 61 98% (91–100) SIT1 + 77 97% (91–100) 73 89% (80–95) 61 59% (46–71) (95–100) sP22-IGRA2 SIT3 77 94% (85–98) 77 94% (85–98) 61 67% (54–79) SIT + 77 97% (91–100) 73 90% (81–96) 61 61% (47–73) cP22-IGRA3 SICCT4 77 91% (82–96) 77 100% 61 80% (68–89) (95–100) SIT + 77 96% (89–99) 73 89% (80–95) 61 66% (52–77) 5 P22-ELISA sP22-IGRA 77 91% (82–96) 74 95% (87–99) 68 84% (73–92) SICCT4 + 77 97% (91–100) 73 95% (87–98) 61 67% (54–79) cP22-IGRA6 77 86% (76–93) 74 96% (89–99) 68 85% (75–93) sP22-IGRA STAND- 77 77% (66–86) 74 100% 68 96% (88–99) SICCT + 77 97% (91–100) 73 96% (88–99) 61 67% (54–79) IGRA7 (95–100) cP22-IGRA 8 DIVA-IGRA 77 71% (60–81) 74 100% 68 100% SICCT + 77 95% (87–99) 73 95% (87–98) 61 79% (66–88) (95–100) (95–100) P22-ELISA P22-ELISA 77 74% (63–83) 74 93% (85–98) 68 96% (88–99) sP22-ST5 + 77 95% (87–99) 73 88% (78–94) 61 82% (70–91) MPB83- 77 75% (64–84) 74 92% (83–97) 68 94% (86–98) sP22-IGRA ELISA sP22-ST + 77 94% (85–98) 73 88% (78–94) 61 93% (84–98) 1 sP22-ST, P22-ELISA single P22 skin test; 2 cP22-ST, comparative P22 skin test; 3 SIT, single intradermal tuberculin test; 4 SICCT, single intradermal cervical comparative tuberculin cP22-ST6 + 77 95% (87–99) 73 95% (87–98) 61 84% (72–92) test; 5 sP22-IGRA, single P22 IGRA test; 6 cP22-IGRA, comparative P22 IGRA test; sP22-IGRA 7 STAND-IGRA, standard tuberculin IGRA test; 8 DIVA-IGRA, differentiating Infected from cP22-ST + 77 94% (85–98) 73 95% (87–98) 61 95% (86–99) Vaccinated animals (ESAT-6/CFP-10 peptide cocktail) IGRA test. 9 Number of animals P22-ELISA tested. 10 Clopper–Pearson 95% confidence interval for Se. 11 Clopper–Pearson 95% confidence interval for Sp. sP22-IGRA + 77 92% (84–97) 74 95% (87–99) 68 84% (73–92) TB, tuberculosis; BCG, bacille Calmette-Guérin; IGRA, interferon-gamma release assay. STAND-IGRA7 P22-ELISA + 77 95% (87–99) 74 89% (80–95) 68 79% (68–88) sP22-IGRA P22 ELISA + 77 95% (87–99) 74 91% (81–96) 68 81% (70–89) cP22-IGRA Interferon-Gamma Release Assays P22-ELISA + 77 87% (77–94) 74 92% (83–97) 68 90% (80–96) The sP22-IGRA showed the highest Se among tests, being a 5, MPB83-ELISA 14, and 20 p.p. more sensitive than the cP22-IGRA, the STAND- P22-ELISA + 77 90% (81–95) 74 93% (85–98) 68 91% (82–97) IGRA, and the DIVA-IGRA, respectively. Indeed, the sP22-IGRA STAND-IGRA detected 12 positive goats more than the STAND-IGRA, without SIT + 77 95% (87–99) 73 93% (85–98) 61 67% (54–79) significant agreement between tests (k = 0.4, p = 0.098) and STAND-IGRA diagnostic results significantly different (Supplementary Data, p SICCT + 77 95% (87–99) 73 100% 61 80% (68–89) = 0.005). The sP22-IGRA and the cP22-IGRA showed similar STAND-IGRA (95–100) specificities in both herds B and C, being a 4–5 p.p. less 1 SIT, single intradermal tuberculin test; 2 sP22-IGRA, single P22 IGRA test; 3 cP22-IGRA, specific than the STAND-IGRA and the DIVA-IGRA. In herd comparative P22 IGRA test; 4 SICCT, single intradermal cervical comparative intradermal C, both cP22-IGRA and sP22-IGRA were a 10–9 p.p. and a tuberculin test; 5 sP22-ST, single P22 skin test; 6 cP22-ST, comparative P22 skin test; 16–15 p.p. less specific than the STAND-IGRA and the DIVA- 7 STAND-IGRA, standard tuberculin IGRA test. 8 Number of animals tested; 9 Clopper– Pearson 95% confidence interval for Se. 10 Clopper–Pearson 95% confidence interval IGRA, respectively. for Sp. TB, tuberculosis; BCG, bacille Calmette-Guérin; IGRA, interferon-gamma release assay. Serological Tests In terms of Sp and Se, diagnostic results of P22-ELISA were Complementarity of Diagnostic Tests similar to diagnostic results of MPB83-ELISA. In herd A, the Combined interpretation of P22-based tests was evaluated. MPB83-ELISA detected 10 TB positive animals more than Results of Sp and Se of complementarity of diagnostic tests are the P22-ELISA, and the P22-ELISA detected nine TB positive shown in Table 4. In general, complementarity between tests animals more than the MPB83-ELISA, and the agreement yielded an overall rise of Se with a variable reduction in the Sp. between tests was considered fair although statistically significant The combination of cP22-ST + P22 ELISA improved the Se in (k = 0.35, p = 0.001). In herd B, diagnostic results of Sp showed 20 p.p. and displayed a similar Sp in both herds B and C, being a moderate but significant agreement between ELISA tests (k = the combined interpretation with the best results in all situations. 0.51, p < 0.001), but in herd C, no agreement was observed (k = The combination of SICCT + P22 ELISA showed similar results −0.05, p = 0.33). of Se and Sp in herd B. In herd C, the latter combination detected Frontiers in Veterinary Science | www.frontiersin.org 4 July 2020 | Volume 7 | Article 374 Arrieta-Villegas et al. P22 Antigen for Diagnosis of TB in Goats 10 false-positives more than the cP22-ST + P22-ELISA, reducing performances by DOR analysis. The performances of tuberculin- its Sp in 16 p.p., and with diagnostic results significantly different based tests were followed by the combinations of SICCT with between tests (p = 0.004). The combination of cP22-ST + cP22- the P22-IGRAs, which increased Se at the cost of a certain loss IGRA improved the Se and Sp in herd B at a similar level than the in Sp. Moreover, the combination of cP22-ST + P22-ELISA combined interpretations above described, but in herd C, the Sp clearly increased the Se with the benefit of a minimal decrease was reduced in 11 p.p. respect to the cP22-ST + P22-ELISA test. of Sp, showing similar results than the combination of SICCT The combination of current diagnostic tests, e.g., SIT and + P22-ELISA. These findings are in concordance with previous SICCT, with other diagnostic tests increased the Se but not the Sp, studies of P22-ELISA and tuberculin-based skin testing. In cattle, except for the SICCT + STAND-IGRA. The latter combination the combination of SIT + P22-ELISA showed an improvement improved the Se in 4 and 18 p.p. compared to the SICCT and of Se of 30 and 6 p.p. compared to the SIT and the P22- the STAND-IGRA alone, respectively, and maintained the Sp ELISA alone, respectively (25). In another study conducted in in herd B but not in herd C (reduction of 16 p.p. compared goats (39), the same combination improved the Se of the SIT to the STAND-IGRA alone). In herd A, the combined results and the P22-ELISA in 19 and 9.5 p.p., respectively. Also, in of MPB83-ELISA + P22-ELISA improved the Se in 12 and 13 the same study in goats, the combination of SICCT + P22- p.p. with respect to the MPB83-ELISA and the P22-ELISA alone, ELISA improved the Se of the SICCT in a 24 p.p. These results respectively, and maintained the Sp in herd B, and in herd confirmed the benefits of the strategic use of serological and C showed a mild reduction of Sp (4 and 6 p.p. of reduction CMI-based diagnostic tests in parallel to maximize the Se in with respect to the MPB83-ELISA and the P22-ELISA alone, infected settings. respectively). Other combinations of tests did not improve the Se In the TB-VAC context, the combination of P22-ELISA with and the Sp, as did the aforementioned combined interpretations. the two P22-based STs showed similar performances than the DIVA-IGRA. However, the latter showed considerably lower Performance of Diagnostic Tests Se than the combinations of P22-ELISA with P22-based STs The results of DOR to assess the diagnostic performance for each (reduction in 23–24 p.p.). Previous studies reported the excellent test are represented in Figure 1. In general, a reduced DOR in Sp (16) and the lack of Se (40) of DIVA-IGRA, although the TB-VAC scenario was observed compared to the conventional DOR analysis tended to overestimate the Sp in this study. The Se one (0.47, 95% CI: 0.28–0.654, of mean reduction in log DOR). of vaccine-associated diagnostic tests is an essential requirement In the conventional context, SICCT + STAND-IGRA (3.38, 95% for the development of an integral vaccination strategy (41), CI: 2.35–4.41), SICCT alone (3.16, 95% CI: 2.36–3.97), SICCT and the combination of cP22-ST + P22-ELISA showed an + cP22-IGRA (2.94, 95% CI: 1.12–4.76), and SICCT + sP22- efficient and innovative diagnostic approach in the TB-VAC IGRA (2.81, 95% CI: 1.08–4.54) showed the best performances context, showing the highest combined Se and Sp values (94 and (Figure 1A). In TB-VAC context, the best performances were 95%, respectively). observed in DIVA IGRA (2.53, 95% CI: 1.98–3.8), cP22-ST + Concerning the use of the ST in solitary, the P22-based STs P22 ELISA (2.44, 95% CI: 0.97–3.92), and sP22-ST + P22 ELISA showed lower Se compared to both the SIT and the SICCT tests, (2.31, 95% CI: 0.95–3.67) (Figure 1B). although previous studies in dairy goat flocks, with larger samples and different epidemiological situations, have shown lower Se for SIT (65%, 95% CI: 63.3–68.2) (8) and SICCT (44.5%, 95% DISCUSSION CI: 35–55) (42). However, the Se of the cP22-ST (74%, 95% CI: 63–83) was similar to Se observed in two previous studies Efficient and accurate diagnosis is of paramount importance for using DIVA STs (based on the peptide cocktails ESAT-6, CFP- the success of eradication programs based on test and slaughter 10, and Rv3616c) developed for the diagnosis of TB in cattle: strategy. Here, the performance of new P22 antigenic complex- 76%, 95% CI: 59–93 (43) and 75%, 95% CI: 47.7–97.7 (44). In based cell-mediated and humoral tests for the diagnosis of TB the latter, the addition of the Rv3020c peptide improved the in goats was assessed under different epidemiological and TB Se to reach 87.5% (95% CI: 61.7–98.5), being similar to the control scenarios. Se of sP22-ST (87%, 95% CI: 74–94) obtained in the present Recently, the P22 antigenic complex has been evaluated for study. On the other hand, in BCG-vaccinated animals, the Sp the detection of IgG in ELISA tests in different species: cattle of SIT and SICCT decreased dramatically (27 and 20 p.p. of goat, sheep, pigs, and wild boar (24–27), red deer (28), badgers reduction, respectively), whereas the Sp of sP22-ST and cP22- (29), and alpacas and llamas (30). In the present study, the ST remained high (97 and 98%, respectively). These findings performance of the P22 antigenic complex for diagnostic tests again highlight the suitability of P22-based STs as TB vaccine- based on CMI, namely, STs and IGRA, was evaluated for the associated diagnostic candidates, although improvements to first time in goats. Indeed, the use of P22 for IGRA tests has increase the Se should be necessary. only been reported in red deer experimentally infected with M. Moreover, herd PTB status and MAP vaccination may also bovis (37). affect the interpretation of the results. MAP infection was not The combined interpretation of tests leads to a substantial reported in farms B and C, and no recent clinical history of improvement of Se at the expense of a variable loss of Sp. PTB was observed by the veterinarians. Despite this, vaccination As expected, in the conventional context, the SICCT alone or against MAP is a common practice in small ruminants in combined with the STAND-IGRA (8, 11, 38) showed the best Spain (12), and diagnostic interferences due to MAP vaccination Frontiers in Veterinary Science | www.frontiersin.org 5 July 2020 | Volume 7 | Article 374 Arrieta-Villegas et al. P22 Antigen for Diagnosis of TB in Goats FIGURE 1 | Diagnostic test performance measured by diagnostic odds ratio (DOR). (A) Conventional (unvaccinated scenario). (B) Tuberculosis (TB)-VAC scenario, animals were vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG). sP22-ST, single P22 skin test; cP22-ST, comparative P22 intradermal skin test; SIT, single intradermal tuberculin test; SICCT, single intradermal cervical comparative tuberculin test; sP22-IGRA, single P22 interferon-gamma release assay (IGRA) test; cP22-IGRA, comparative P22 IGRA test; STAND-IGRA, standard tuberculin IGRA test; DIVA-IGRA, differentiating infected from vaccinated animals (ESAT-6/CFP-10 peptide cocktail) IGRA test. on TB diagnosis cannot be ruled out in these two MAP- fact that P22 complex contains 21 proteins also present in vaccinated herds. In this sense, strong reactions to PPD-A were M. avium (23), which can cause cross-reactivity with MAP observed at skin testing (Supplementary Data), but the results vaccination and/or infection. Indeed, the interference of MAP of comparative tests (cP22-ST and SICCT) showed higher Sp vaccination on STAND-IGRA has been previously observed in compared to their respective single STs (i.e., sP22-ST and SIT). adult MAP-vaccinated goats (13, 14, 45). The Sp of P22-IGRAs These findings indicate that some degree of cross-reactivity due considerably decreased in BCG-vaccinated herds compared to to MAP vaccination was still maintained. Similarly, interferences that previously described for the STAND-IGRA (16). Overall, of MAP vaccination on TB diagnosis, mainly in CMI-based the results of sP22-IGRA suggest that this test could be a diagnostic tests, were previously observed in MAP-vaccinated potentially valuable tool for TB eradication in endemic areas, goats (14, 45). although further studies to determine the optimal concentration Surprisingly, the P22-based IGRAs, particularly the sP22- of P22 are required to improve its Sp with a minimal IGRA, showed higher Se compared to STAND-IGRA and even loss of Se. higher compared to DIVA-IGRA. However, the Se of sP22-IGRA Serological diagnostics is a cost-effective alternative for was similar to that previously observed by the STAND-IGRA TB diagnostics. However, the Se of antibody-based diagnostic (92%, 95% CI: 84–96) in other studies conducted in goats (26). tests was generally lower compared to tests based on CMI The results of Se of the cP22-IGRA in the present study were (46, 47). In the present study, the Se of P22-ELISA was also similar to those previously observed in experimentally M. slightly lower than that in previous studies in goats and bovis-infected red deer (37). However, a slight loss of Sp in cattle (25, 39). This loss of Sp might be explained by the the P22-IGRAs was detected compared to STAND-IGRA. Even fact that animals from herd A were not vaccinated against so, the Sp was within ranges (95–100%) described for STAND- MAP nor subjected to frequent intradermal testing, factors that IGRA in previous studies (11, 38, 45). This mild reduction could enhance humoral responses against MTBC antigens (48). in Sp could be explained by the high concentration of P22 Interestingly, the Se was significantly enhanced when using P22 used for stimulation of whole blood (20 µg/ml) and by the and MPB83 ELISAs in parallel. Thus, even though MPB83 is Frontiers in Veterinary Science | www.frontiersin.org 6 July 2020 | Volume 7 | Article 374 Arrieta-Villegas et al. P22 Antigen for Diagnosis of TB in Goats a major component of the P22 complex, specific IgGs of some ETHICS STATEMENT infected animals were only detectable using the MPB83 purified recombinant protein alone, while others were only detected All animals included in this study belonged to commercial using the P22 complex, which contains additional serodominant farms and were not experimental animals. All sampling and epitopes (23). handling procedures were carried out by authorized veterinarians Finally, Sp of the P22-ELISA reached considerably higher according to standard farm methods and in conformity with Sp in MAP-vaccinated (and BCG-unvaccinated, i.e., herd B) Spanish legislation (Royal Decree 2611/1996 and amendments) goats (93%) compared to that previously found in Spanish and European Union laws for the protection of animals used (78%) and Norwegian MAP-vaccinated goats (58%) (24). In the for scientific purposes (2010/63/EU). Test and slaughter of latter study, besides MAP vaccination, MAP coinfection and/or positive animals, as well as post-mortem sampling to confirm the contact with environmental mycobacteria was not discarded as disease, were conducted according to the regulations defined by a source of diagnostic interference. Interestingly, in the present the Catalan Government (Resolution AAM/1314/2014). Written study, the Sp was also high in BCG- and MAP-vaccinated goats informed consent was obtained from the owners for the (96%), suggesting that BCG vaccination does not induce antibody participation of their animals in this study. responses that cause interference on the diagnosis by the P22- ELISA. The absence of antibody responses was consistent with AUTHOR CONTRIBUTIONS the fact that the BCG Danish strain used for vaccination expresses low levels of MPB83 and MPB70 (49), which are the most BP and JB contributed to conceptualization. CA-V, BP, and MG abundant proteins of the P22 antigenic complex (23). Moreover, contributed to data curation. CA-V and BP performed the formal tuberculin skin testing after 42 days of MAP or BCG vaccination analysis. BP and LJ acquired funding. CA-V, BP, MG, EV, MD, caused a boosting effect on humoral responses against tuberculin IM, JI-L, and JB contributed to the investigation. CA-V, BP, JI- antigens, resulting in false-positive cattle for an MPB83-based L, EV, and MD contributed to the methodology. BP and LJ ELISA (50). Here, minimal or no boosting effects of MAP/BCG contributed to project administration. JI-L and MS acquired vaccination due to skin testing were observed on the P22-ELISA. resources. CA-V and BP wrote the original draft. JB, LJ, JI-L, EV, Indeed, goats from herd B were sampled around 2 years after and MD contributed to writing, reviewing, and editing. vaccination against MAP, and ST was performed once or twice after MAP vaccination. Also, 34/68 goats from herd C were FUNDING vaccinated with BCG and Gudair R at 9–10 months before the sampling, whereas the rest of the animals were vaccinated more This work was funded by a grant from Instituto Nacional than 1 year before, and no ST was performed since. Based on de Investigación y Tecnología Agraria y Alimentaria (INIA), the results herein, the P22-ELISA seemed to be a useful ancillary reference number RTA2015-00043-C02-00 (FEDER co-funded). diagnostic tool, either in BCG or MAP vaccination context, IRTA is supported by CERCA Program/Generalitat de Catalunya. although it should be confirmed in further studies with larger Claudia Arrieta-Villegas is recipient of a PhD grant from INIA sized herds. (FPI-INIA 2016: CPD2016-0109) funded by the Spanish Ministry In conclusion, this study reinforces the applicability of the of Economy and Competitiveness. P22 antigen complex as a complementary instrument for TB diagnostics in goats under different control scenarios. The ACKNOWLEDGMENTS P22 serological diagnostic is a cost-effective alternative, and combined interpretation with STs, either with PPD-B or P22, We are grateful to Maite Martín, Zoraida Cervera, Carlos Lopez- showed promising results. Moreover, the use of P22 antigenic Figueroa, and the staff of Field Studies Unit of IRTA-CReSA for complex in CMI-based diagnostic tests showed encouraging their technical support. results, being suitable for further research on the improvement of TB diagnostics. SUPPLEMENTARY MATERIAL DATA AVAILABILITY STATEMENT The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets. All datasets generated for this study are included in the 2020.00374/full#supplementary-material Supplementary Material. Supplementary Data | Raw data diagnostic tests. REFERENCES 2. Napp S, Allepuz A, Mercader I, Nofrarías M, López-Soria S, Domingo M, et al. 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Conflict of Interest: The authors declare that the research was conducted in the A defined antigen skin test for the diagnosis of bovine tuberculosis. Sci Adv. absence of any commercial or financial relationships that could be construed as a (2019) 5:eaax4899. doi: 10.1126/sciadv.aax4899 potential conflict of interest. 44. Jones GJ, Whelan A, Clifford D, Coad M, Vordermeier HM. Improved skin test for differential diagnosis of bovine tuberculosis by the addition Copyright © 2020 Arrieta-Villegas, Infantes-Lorenzo, Bezos, Grasa, Vidal, Mercader, of Rv3020c-derived peptides. Clin Vaccine Immunol. (2012) 19:620– Singh, Domingo, de Juan and Pérez de Val. This is an open-access article distributed 2. doi: 10.1128/CVI.00024-12 under the terms of the Creative Commons Attribution License (CC BY). The use, 45. Roy Á, Infantes-Lorenzo JA, Blázquez JC, Venteo Á, Mayoral FJ, Domínguez distribution or reproduction in other forums is permitted, provided the original M, et al. Temporal analysis of the interference caused by paratuberculosis author(s) and the copyright owner(s) are credited and that the original publication vaccination on the tuberculosis diagnostic tests in goats. Prev Vet Med. (2018) in this journal is cited, in accordance with accepted academic practice. No use, 156:68–75. doi: 10.1016/j.prevetmed.2018.05.010 distribution or reproduction is permitted which does not comply with these terms. Frontiers in Veterinary Science | www.frontiersin.org 9 July 2020 | Volume 7 | Article 374