TYPE Original Research PUBLISHED 10 November 2022 DOI 10.3389/fvets.2022.1042428 Effect of a recent parenteral OPEN ACCESS dexamethasone and ketoprofen administration on the EDITED BY Jesus Hernandez, Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico REVIEWED BY immunological diagnosis of tuberculosis in goats Saurabh Gupta, GLA University, India Raul G. Barletta, University of Nebraska-Lincoln, United States Javier Ortega1,2 , Lucia de Juan1,2 , Iker A. Sevilla3 , *CORRESPONDENCE Javier Bezos Joseba M. Garrido3 , Álvaro Roy2 , Carlos Velasco2 , jbezosga@visavet.ucm.es Beatriz Romero1,2 , Mercedes Domínguez4 , SPECIALTY SECTION This article was submitted to Bernat Pérez de Val5 , Carolina Nebot6 , Veterinary Infectious Diseases, a section of the journal José Luis Sáez-Llorente7 , Julio Álvarez1,2 and Javier Bezos1,2* Frontiers in Veterinary Science 1 Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, RECEIVED 12 September 2022 Madrid, Spain, 2 VISAVET Health Surveillance Centre, Complutense University of Madrid, Madrid, ACCEPTED 28 October 2022 Spain, 3 Animal Health Department, NEIKER-Basque Institute for Agricultural Research and PUBLISHED 10 November 2022 Development, Basque Research and Technology Alliance (BRTA), Derio, Spain, 4 Unidad de Inmunología Microbiana, Centro Nacional de Microbiología, Instituto de Investigación Carlos III, CITATION Madrid, Spain, 5 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Universitat Ortega J, de Juan L, Sevilla IA, Autònoma de Barcelona, Barcelona, Spain, 6 Laboratorio de Higiene, Inspección y Control de Garrido JM, Roy Á, Velasco C, Alimentos (LHICA), Facultad de Veterinaria, de Santiago de Compostela (Campus de Lugo), Lugo, Romero B, Domínguez M, Pérez de Spain, 7 Ministerio de Agricultura, Pesca y Alimentación, Madrid, Spain Val B, Nebot C, Sáez-Llorente JL, Álvarez J and Bezos J (2022) Effect of a recent parenteral dexamethasone and ketoprofen administration on the Caprine tuberculosis (TB) is a zoonosis caused by members of the immunological diagnosis of Mycobacterium tuberculosis complex (MTBC). Caprine TB eradication tuberculosis in goats. Front. Vet. Sci. 9:1042428. programmes are based mainly on intradermal tuberculin tests and doi: 10.3389/fvets.2022.1042428 slaughterhouse surveillance. Different factors may affect the performance COPYRIGHT of the TB diagnostic tests used in caprine herds and, therefore, their © 2022 Ortega, de Juan, Sevilla, ability to detect infected animals. The present study evaluates the effect Garrido, Roy, Velasco, Romero, Domínguez, Pérez de Val, Nebot, of the fraudulent administration of two anti-inflammatory substances, Sáez-Llorente, Álvarez and Bezos. This dexamethasone and ketoprofen, on the performance of the TB diagnostic is an open-access article distributed under the terms of the Creative techniques used in goats, as well as the suitability of high performance Commons Attribution License (CC BY). liquid chromatography (HPLC) for their detection in hair samples. The The use, distribution or reproduction animals (n = 90) were distributed in three groups: (1) a group treated with in other forums is permitted, provided the original author(s) and the copyright dexamethasone (n = 30); a second group treated with ketoprofen (n = 30); owner(s) are credited and that the and a third non-treated control group (n = 30). Both dexamethasone original publication in this journal is and ketoprofen groups were subjected to intramuscular inoculation cited, in accordance with accepted academic practice. No use, distribution with the substances 48 h after the administration of bovine and avian or reproduction is permitted which purified protein derivatives (PPDs), that is, 24 h before the tests were does not comply with these terms. interpreted. All the animals were subjected to the single and comparative intradermal tuberculin (SIT and CIT, respectively) tests, interferon-gamma release assay (IGRA) and P22 ELISA. The number of SIT test reactors was significantly lower in the dexamethasone (p = 0.001) and ketoprofen (p < 0.001) groups 72 h after the bovine PPD inoculation compared with the control group. A significantly higher number of positive reactors to IGRA was detected within the dexamethasone group (p = 0.016) 72 h after PPD administration compared to the control group. Dexamethasone Frontiers in Veterinary Science 01 frontiersin.org Ortega et al. 10.3389/fvets.2022.1042428 and ketoprofen detection in either hair or serum samples was challenging when using HPLC since these substances were not detected in animals whose skin fold thickness (SFT) was reduced, what could be an issue if they are used for fraudulent purposes. In conclusion, the parenteral administration of dexamethasone or ketoprofen 48 h after the PPDs administration can significantly reduce the increase in SFT (mm) and subsequently the number of positive reactors to SIT test. KEYWORDS caprine tuberculosis, dexamethasone, ketoprofen, diagnosis, intradermal tuberculin test Introduction of betamethasone (16). Nevertheless, previous studies have shown that the parenteral administration of dexamethasone Caprine tuberculosis (TB) is a chronic zoonotic disease reduces IFN-γ production in TB-infected cattle, resulting in caused by members of the Mycobacterium tuberculosis complex false negative reactors to the IGRA (17, 18). These results (MTBC), and principally Mycobacterium tuberculosis variant therefore suggest that the administration of corticosteroids bovis (M. bovis) and M. tuberculosis variant caprae (M. caprae), can interfere with the diagnosis of TB in ruminants in being M. caprae the main causative agent in Spain (1). different ways depending on the administration route (16–18). Tuberculosis is known to be a multi-host disease that affects a The parenteral administration of anti-inflammatory substances wide range of domestic and wildlife species and humans (2–5). could potentially be used with fraudulent purposes in order Moreover, the presence of TB in livestock leads to economic to interfere with the diagnosis of TB by inhibiting the losses owing to the reduction in milk yield and fertility, local (SIT/CIT tests) and systemic (IGRA) inflammatory condemnations at slaughterhouses, restrictions on markets and reactions in goats. However, to the best of the authors’ the cost of diagnosis (6–8). In the case of Spain, whose caprine knowledge, no previous studies have evaluated the effect of population is one of the largest in the European Union (EU) the parenteral administration of anti-inflammatory substances (9), certain regions have implemented regional eradication on the outcome of the TB diagnostic tests in goats. In fact, programmes (10). These caprine TB eradication programmes not only corticosteroids, but also other anti-inflammatory are based mainly on test and cull strategies using the single and substances could be used for this purpose according to personal comparative intradermal tuberculin (SIT and CIT, respectively) communication of official veterinary services of Spain. In tests as the cornerstone of the ante-mortem diagnosis, and this context, in the recent years the authorities of different slaughterhouse surveillance (6). The interferon-gamma release regions in Spain have started to research the fraudulent use assay (IGRA) has, in specific cases, also been used to diagnose of different substances parenterally administered in order to TB in goats (6, 10, 11). Moreover, the prior diagnosis of TB in modify the result of official TB diagnostic tests in ruminants (19). ruminants is considered a requirement for movements between Moreover, it is important to highlight the difficulties involved in Member States by the current European legislation (Regulation demonstrating this type of fraudulent activities due to the high EU 2016/429). This legislation also considers the IGRA as an variety of substances and administration protocols which can be official test to allow movements of animals within the EU. applied in ruminants to modify the TB diagnostic tests outcome. Both of these techniques, which are based on the cellular The development and evaluation of methodologies, in order to immune response, are valuable diagnostic tools, although they establish effective protocols for the detection of these substances, are not perfect in terms of sensitivity and specificity, and are consequently of paramount importance. Therefore, the aim several factors could affect their performance (11–15). One of the present study was to determine, for the first time, whether of these factors is the administration of anti-inflammatory the parenteral administration of dexamethasone or ketoprofen substances, which are, according to previous studies in goats 48 h after the PPDs administration interferes with the diagnosis (16) and cattle (17, 18), able to modify the results of official of caprine TB by intradermal tests, and whether this protocol TB diagnostic tests carried out in ruminants. A recent study also affects the results of other diagnostic techniques such as in goats demonstrated that a recent topical application of a IGRA and Ab-based tests. A specific protocol for the detection corticosteroid (betamethasone) at the PPD injection site can of dexamethasone and ketoprofen in serum and hair samples significantly reduce the number of reactors to SIT/CIT tests (16). was also developed and applied in order to demonstrate the This effect was not observed on the IGRA and P22 ELISA results, presence of these substances in the goats from the different and this finding was associated with the topical application experimental groups. Frontiers in Veterinary Science 02 frontiersin.org Ortega et al. 10.3389/fvets.2022.1042428 Materials and methods and ratified by the local authority (PROEX11/18; Comunidad de Madrid). Experimental design The study was carried out in a TB-infected dairy goat Intradermal tuberculin tests herd of the Guadarrama breed located in central Spain. This herd (n = 161) had previously been confirmed as TB-infected The animals were subjected to SIT and CIT tests, which by means of bacteriological culture (M. bovis SB0121). The were carried out by intradermally inoculating them with 0.1 ml animals were subjected to SIT test, CIT test, IGRA (in plasma of bovine and avian PPDs (CZ Vaccines, Porriño, Spain) using samples) and P22 ELISA (in serum samples) 3 months before a Dermojet syringe (AkraDermojet, France) on the left-medial the start of this study and there was an apparent prevalence and right-medial side of the neck, respectively. The intradermal of 40.9% (95% CI 33.7–48.7), 7.5% (95% CI 4.3–12.6), 50.3% inoculation of PPDs were carried out at day 0 in all groups and (95% CI 42.7–57.9) and 43.5% (95% CI 36.1–51.2), respectively. the reactions were interpreted 48 h (day 2) and 72 h (day 3) According to these results, this herd was considered a high later by the same veterinarian (Figure 1). SIT and CIT tests were prevalence herd. Furthermore, this herd had undergone a performed according to the protocol published by the European vaccination programme against Mycobacterium avium subsp. Union Reference Laboratory (EU-RL) for bovine TB following paratuberculosis (MAP), using the Gudair vaccine (CZ Vaccines, Regulation EU 2016/429, the Commission Delegated Regulation Porriño, Spain) in 6-months old animals. The goats were EU 2020/688 and Royal Decree 2611/1996. The interpretation of selected on the basis of previous TB testing results using results was carried out as described previously (20). SIT test and IGRA in parallel. The animals (n = 90) were randomly distributed in three experimental groups: (1) treated with dexamethasone (n = 30); (2) treated with ketoprofen (n = 30) and (3) control (n = 30) groups (Figure 1). All the Interferon-gamma release assay animals were subjected to the SIT and CIT tests by means of the intradermal injection of bovine and avian PPDs (day 0), and Blood samples were collected from the jugular vein by two readings were conducted: one at day 2 (48 h after the PPDs venepuncture using evacuated tubes (BD Vacutainer Becton, injections) and the official reading at day 3 (72 h after the PPDs Dickinson and Company, Franklin Lakes, USA) with lithium injections). Just after the first reading, the recommended doses heparin at days 0 and 3 for the detection of IFN-γ production (in a final volume of 1.5 ml) of dexamethasone (Dexamethasone (Figure 1). In the laboratory, the blood samples were stimulated Sodium Phosphate 2 mg/ml, Cenmetasona, CENASIVA S.L., with bovine and avian PPDs (CZ Vaccines, Porriño, Spain) Reus, Spain) or ketoprofen (Ketoprofen 100 mg/ml, Ketink, at a final concentration of 20 µg/ml each and were processed Industrial Veterinaria S.A., Barcelona, Spain) were administered as described elsewhere (11). IFN-γ production in plasma intramuscularly on the left side of the neck in the dexamethasone was measured using a commercial kit (Bovigam TB, Thermo and ketoprofen groups, respectively (Figure 1). IGRA and P22 Fisher Scientific, Waltham, USA) following the manufacturer’s ELISA were performed using plasma and serum, respectively, instructions, and the results were interpreted as described both obtained from whole blood samples collected at days 0 and previously (21). 3 (Figure 1). Moreover, hair and serum samples were collected at day 3 (Figure 1) in order to detect dexamethasone and ketoprofen using High Performance Liquid Chromatography- P22 ELISA Mass Spectrometry (HPLC-MS). Razor blades were used to collect fifteen hair samples from the left and right sides of Specific antibodies against the MTBC were detected using the neck and another 15 from the left and right sides of the an in-house indirect enzyme-linked immunosorbent assay based backs of the animals from the dexamethasone, ketoprofen and on the multiprotein complex P22 (P22 ELISA) obtained by control groups. Serum samples were collected from all the the inmunopurification of bovine PPD (CZ Vaccines, Porriño, animals in each experimental group. In addition, serum samples Spain). The bovine and avian PPDs traditionally used for from 10 animals in each group were assayed for the analysis TB diagnosis in ruminants contain 456 and 1019 proteins of the cytokine/chemokine production pattern in the three respectively, reduced to 118 in P22. There are 143 proteins experimental groups. that are present both in bovine and avian PPDs, potentially All handling, testing and sampling procedures were responsible of the cross reactivity, which are reduced to 43 in the carried out by qualified veterinarians in compliance P22 complex, explaining the higher specificity obtained using with European (86/609/CEE) and Spanish (RD 53/2013) P22 (22). Blood samples were collected from the jugular vein legislation. The procedures employed in the current study using plastic serum tubes (BBD Vacutainer Becton, Dickinson were similarly approved by an institutional ethical committee and Company, Franklin Lakes, USA) at days 0 and 3 (Figure 1). Frontiers in Veterinary Science 03 frontiersin.org Ortega et al. 10.3389/fvets.2022.1042428 FIGURE 1 Summary of the experimental design. White silhouettes represent goats untreated and black goat silhouettes represent goats treated with dexamethasone (0.06 mg/kg) or ketoprofen (3 mg/kg). The ELISA was performed on these serum samples as described were mixed several times with methanol to obtain a working previously (23). The serum sample results were expressed standard solution mixture of the selected drugs at 1 and as an ELISA percentage (E%), calculated according to the 0.1 µg/ml. following formula: The serum and hair samples of the dexamethasone and Sample E% = [mean sample OD / (2 x mean of negative ketoprofen groups were analyzed in an LC-MS/MS system control OD)] × 100 (Bruker Bremen, Germany). Analytes were separated on an Serum samples with E% values >150 or 100 were considered Acquity UPLC BEH C18 chromatographic column (2.1 × positive to the standard and severe interpretations, respectively, 100 mm, 1.7 µm) obtained from Waters (Milford, USA) for as described elsewhere (20, 23). dexamethasone detection and on an Intensity Solo HPLC column C18 (2.0 × 100 mm, 2 µm) for ketoprofen, also from Bruker Daltonik. The separation of the analytes was achieved for both substances in a gradient mode phase A High performance liquid mixture (0.1% acidified water with formic acid) and a phase chromatography-mass spectrometry B mixture (methanol 0.1% acidified with formic acid for ketoprofen and acetonitrile 0.1% acidified with formic acid Dexamethasone (purity > 98%), ketoprofen (purity > 99%), for dexamethasone). formic acid (purity > 99% for analysis) and sodium acetate (purity > 98% for analysis) were purchased from Sigma- Aldrich (St. Louis, MO, USA), while the different organic HPLC-MS in serum samples solvents (HPLC grade) were obtained from Merk (Darmstadt, Five hundred µl of serum sample was transferred Germany). The water employed in the mobile phase and for into 2 ml disposable Eppendorf tubes, and 1.5 ml of the sodium solution 1M was obtained from Milli-Q water from acetonitrile (ketoprofen) and 800 µl of acetonitrile Millipore system (Bedford, MA, USA). Nitrogen generated by a (dexamethasone) (Merk, Darmstadt, Germany) were nitrogen generator obtained from Peak Scientific Instruments, added. Analyte-free goat serum samples were spiked with Ltd. (Chicago, IL, USA) was employed for MS detection ketoprofen to 20, 50, 100, 150 and 300 ng/ml or with and solvent evaporation. Stock solutions of dexamethasone dexamethasone to 1, 5, 10, 25, 50 and 100 ng/ml for (0.8 mg/mL) and ketoprofen (0.8 mg/ml) were prepared in quantification. The limit of detection was 10 ng/sample methanol (Merk, Darmstadt, Germany) and stored in the for the detection of dexamethasone and ketoprofen in dark at −18◦ C. These stock solutions of each substance serum samples. Frontiers in Veterinary Science 04 frontiersin.org Ortega et al. 10.3389/fvets.2022.1042428 HPLC-MS in hair samples the experimental group (dexamethasone/ketoprofen/control as The ketoprofen and dexamethasone were extracted as reference category) as the predictor. Quantitative differences in described previously for betamethasone (16). Fifteen µl of the the increase in skin fold thickness (SFT), optical density (OD) extract was then injected into the HPLC column for analysis and E% between days 0, 2 and 3 were analyzed by means of by means of HPLC-MS/MS. Each day of analysis, calibration the Wilcoxon signed-rank test. Quantitative values, such as the curves were prepared with 5 ml of acetonitrile in 15 ml Falcon increase in the SFT (expressed in mm), IFN-γ levels (OD), tubes, to which different amounts of ketoprofen were added ELISA percentage (E%) and the amount of cytokine/chemokines in order to obtain a final concentration of 0.1, 0.2, 2, 5, and (expressed in pg/ml) in the goats in the different groups at 10 ng/ml. In the case of dexamethasone, in order to quantify a given sampling point were compared using the Kruskal- the pharmaceutical, each day of analysis, buffer solutions were Wallis test followed by pairwise tests for multiple comparisons spiked with dexamethasone to final concentrations of 0.1, 0.5, 1, of mean rank sums after adjusting the p-value using the 20, 40, and 80 ng/ml, the extraction protocol was applied, and Bonferroni correction. With regard to IFN-γ levels, Spearman’s the peak area recorded. Equally to serum samples, the limit of rank correlation coefficient (rs ) was used to assess the detection was 10 ng/sample of both substances in hair samples. relationship between ODs in plasma and the pg/ml of cytokine observed in serum samples. Heatmap clustering analysis of cytokine/chemokines in serum samples was conducted using an Cytokine/chemokine production pattern online tool (Heatmapper, 164 www.heatmapper.ca) (26). detection A Luminex-based cytokine/chemokine array detecting 15 Results cytokines and chemokines was applied to the serum samples collected at day 3 from 10 goats randomly selected from Cell-based diagnostic tests each group. The cytokines/chemokines present in the serum samples were then quantified using a bovine multiplex assay kit The number and percentage of positive reactors obtained (MILLIPLEX MAP Kit Bovine Cytokine/Chemokine Magnetic using the different diagnostic tests are summarized in Table 1. Bead Panel 1, 96-Well Plate Assay, Merck Millipore, UK). Serum While in day 2 (before the inoculation of the drugs) there was samples were centrifuged at 10,000 g for 5 min. The serum was no significant (p = 0.27) association between the group and subsequently diluted (1:2 in the assay buffer provided in the the test result, the probability of finding a SIT test reactor was kit). Twenty-five µl of each mix (serum and buffer) were then significantly lower (p < 0.001) in the dexamethasone (OR = analyzed in duplicate on Bio-Plex R 200 (Bio-Rad Laboratories, 0.11, 95% CI 0.02–0.40) and ketoprofen (OR = 0.08, 95% CI S.A., Alcobendas, Spain) in order to determine the amount 0.01–0.30) groups than in the control group at day 3 after (expressed in pg/ml) of IFN-γ, IL-1α, IL-1β, IL-6, IL-8, IL- the bovine PPD inoculation. This finding was due to a lower 17A, IL-36RA, IP-10, MCP-1, MIP-1α MIP-1β, TNFα VEGF-A increase in the SFT in the dexamethasone (p = 0.005) and (proinflammatory response), IL-4 and IL-10 (anti-inflammatory ketoprofen (p < 0.001) groups when compared to the control response) in the serum samples. This result was the mean of two group (Figure 2A). analyses of each sample and multiplied by the dilution factor. When measures obtained in day 2 (48 h, time of application) and 3 (72 h, time of SIT test interpretation) for each experimental group were compared, the SFT at the bovine Statistical analysis inoculation site was reduced in both dexamethasone and ketoprofen groups (Figure 2B). This reduction in the SFT led to Data analyses were performed using SPSS Statistics 25 a significant (p < 0.001) decrease in both the number of reactors (IBM, New York, NY, USA), and R version 4.0.5 software at day 3 respected to those observed at day 2 using standard (24) and interpreted by using a p-value of 0.05 to determine interpretation of the SIT test (Table 1, Figure 3). Interestingly, statistical significance. Wilson’s 95% confidence intervals (95% the reductions in the SFT at the avian PPD inoculation site were CI) were calculated for the percentage of reactors to the not significant in the dexamethasone (p = 0.085) and ketoprofen different diagnostic techniques using WinPepi version 11.6 (25). (p = 0.05) groups between day 2 and day 3 (Figure 2C). The comparison of the proportions of test reactors within a In contrast, a significant increase in the SFT at the bovine given group between days 0 and 3 (IGRA and P22 ELISA) (p = 0.002) and avian (p = 0.01) PPD inoculation site between and 2 and 3 (intradermal tuberculin tests) was performed by day 2 and day 3 was observed in the control group. Moreover, using McNemar’s test. The comparison of the proportions of the increase of the SFT was significantly higher at the bovine test reactors among the different groups at a given sampling PPD inoculation site compared to the avian PPD inoculation point was performed using a logistic regression model with site in the dexamethasone (p = 0.01) and ketoprofen (p = 0.006) the test result (positive/negative) as the outcome variable and groups. In this line, the reduction in the number of reactors to Frontiers in Veterinary Science 05 frontiersin.org Frontiers in Veterinary Science Ortega et al. TABLE 1 Number of positive reactors (including percentage and Wilson’s CI 95%) to diagnostic tests in each experimental group. Day 0 (N, %e ) Day 2 (N, %e ) Day 3 (N, %e ) Group n IGRA IGRA P22 P22 SIT testc CIT testd SIT testc CIT testd IGRA IGRA P22 P22 0.05a 0.1a 100b 150b 0.05a 0.1a 100b 150b Standard Severe Standard Severe Standard Severe Standard Severe Dexamethasone 30 5, 16.7 3, 10.0 13, 43.3 11, 36.7 28, 93.3 30, 100 7, 23.3 21, 70.0 15, 50.0 24, 80.0 5, 16.7 13, 43.3 12, 40.0 8, 26.7 16, 53.3 14, 46.7 (7.3– (3.5– (27.4– (21.8– (78.7– (88.6– (11.8– (52.1– (33.2– (62.7– (7.3– (27.4– (24.6– (14.2– (36.1– (30.2– 33.5) 25.6) 60.8) 54.5) 98.2) 100) 40.9) 83.3) 66.8) 90.5) 33.5) 60.8) 57.7) 44.5) 69.8) 63.9) Ketoprofen 30 7, 23.3 3, 10.0 14, 46.7 12, 40.0 27, 90.0 30, 100 11, 21, 70.0 13, 43.3 17, 56.7 6, 20.0 14, 46.7 8, 26.7 4, 13.3 15, 50.0 13, 43.3 (11.8– (3.5– (30.2– (24.6– (74.4– (88.6– 36.7(21.8– (52.1– (27.4– (39.2– (9.5– (30.2– (14.2– (5.3– (33.2– (27.4– 40.9) 25.6) 63.9) 57.7) 96.5) 100) 54.5) 83.3) 60.8) 72.6) 37.3) 63.9) 44.5) 29.7) 66.8) 60.8) Control 30 10, 33.3 6, 20.0 16, 53.3 15, 50.0 24, 80.0 27, 90.0 5, 16.7 20, 27, 90.0 30, 100 2, 6.6 15, 50.0 13, 43.3 9, 30.0 19, 63.3 16, 53.3 (19.2– (9.5– (36.1– (33.2– (62.7– (74.4– (7.3– 66.7(48.8– (74.4– (88.6– (1.8– (33.2– (27.4– (16.7– (45.5– (36.1– 51.2) 37.3) 69.8) 66.8) 90.5) 96.5) 33.5) 80.7) 96.5) 100) 21.3) 66.8) 60.8) 47.9) 78.1) 69.8) a An animal was considered positive to the IGRA if the optical density (OD) of a sample stimulated with bovine PPD minus the OD of PBS was >0.1 (standard interpretation) or 0.05 (severe interpretation) and greater than the OD of the sample stimulated with avian PPD. b An animal was considered positive to P22 ELISA when the E% value was >150 (standard interpretation) or 100 (severe interpretation). 06 c An animal was considered a positive reactor to the SIT test when there was an increase of ≥ 4 mm (standard interpretation) or >2 mm (severe interpretation) in the skin fold thickness and/or the presence of clinical signs was observed. d An animal was considered a positive reactor to the CIT test when the bovine reaction was greater than the avian reaction by more than 4 mm (standard interpretation) or the bovine reaction was ≥3 mm and greater than the avian reaction (severe interpretation); and/or there were clinical signs at the bovine PPD inoculation site. e Wilson’s 95% confidence intervals. 10.3389/fvets.2022.1042428 frontiersin.org Ortega et al. 10.3389/fvets.2022.1042428 FIGURE 2 Summary of the differences among median skin fold thickness (mm) after bovine PPD injection in the dexamethasone, ketoprofen and control groups at day 3 (A), and after bovine (B) and avian (C) PPD injection in the dexamethasone and ketoprofen groups at 48 h and 72 h. *p = 0.005, **p < 0.001. Frontiers in Veterinary Science 07 frontiersin.org Ortega et al. 10.3389/fvets.2022.1042428 FIGURE 3 Percentage of animals that were positive (gray) and negative (white) to SIT test in the dexamethasone, ketoprofen and control groups at day 2 and day 3. the CIT test was not significant in the dexamethasone (p = 0.50), ELISA, since the differences between the groups in terms of and ketoprofen (p = 0.06) groups between day 2 and day 3 when quantitative (E%) and qualitative (positive reactors) were not using the standard interpretation of the CIT test (Table 1). The significant at day 3 (p = 0.52 and p = 0.55, respectively). In this increase in the SFT was significantly (p < 0.001) higher at bovine respect, a significant increase in the E% was observed between PPD inoculation site compared to those reactions observed at day 0 and day 3 in the dexamethasone (p < 0.001), ketoprofen the avian PPD inoculation site in the reactors to CIT test when (p = 0.001) and control (p < 0.001) groups (Figure 4B). using standard interpretation 48 h after the PPDs administration However, this significant increase in the E% did not lead to in the three groups. a significant increase in the number of positive goats between The intramuscular injection of dexamethasone and day 0 and day 3 using standard (p = 0.25, p = 1 and p = 1 ketoprofen was not associated with the IGRA results, since no in dexamethasone, ketoprofen and control groups, respectively) significant (p = 0.35) association between the group and the or severe interpretation (p = 0.25, p = 1 and p = 0.25 in test result was observed (dexamethasone: OR = 0.87, 95% CI dexamethasone, ketoprofen and control groups, respectively). 0.31–2.44; ketoprofen: OR = 0.47, 95% CI 0.15–1.38) in day 3 using the control group as a reference. Similarly, the differences between the IFN-γ results (OD) at day 3 in the groups were Cytokine/chemokine production pattern not significant (p = 0.47, Figure 4A). A significantly higher detection number of IGRA positive reactors (p = 0.01) was detected within the dexamethasone group when the test was performed A similar pattern was observed for the 15 cytokines and at day 3 when compared to day 0 (Table 1). With regard to chemokines measured on day 3 (Figure 5). No significant the quantitative results, a significant increase (p = 0.02) in the differences (p > 0.05) among the different groups were observed IFN-γ measures was observed between day 0 and day 3 in the in terms of the amount (pg/ml) of each cytokine/chemokine in dexamethasone group. However, no significant differences in the serum samples. Twelve out of the fifteen cytokines analyzed the IGRA results (ODs) were observed between day 0 and day 3 were detected in all animals, regardless of the group. However, in the control (p = 0.58) and ketoprofen (p = 0.06) groups. IL-1β was detected in 9 out of 30 goats, whereas IL-17A and MIP-1β were detected in 16 animals. Nevertheless, this lack of detection was not associated with a specific group. In contrast, Antibody-based diagnostic test high levels of IL-8, IP-10 and TNFα were observed in serum samples taken from animals from all the groups (Figure 5), thus The recent intramuscular administration of dexamethasone suggesting a stronger basal proinflammatory response to the TB or ketoprofen was not associated with the result in the P22 infection in these animals, regardless of the group. Finally, the Frontiers in Veterinary Science 08 frontiersin.org Ortega et al. 10.3389/fvets.2022.1042428 FIGURE 4 Interferon-gamma release assay (IGRA) values expressed as optical density (OD) in the dexamethasone, ketoprofen and control groups at day 0 and day 3 (A) and P22 ELISA values expressed as ELISA percentage (E%) in the dexamethasone, ketoprofen and control groups at day 0 and day 3 (B). correlation (Spearman’s rank correlation coefficient) between effect of the administration of a recent parenteral corticosteroids the amount of IFN-γ in plasma samples observed after the and non-steroid anti-inflammatory substances on different bovine PPD stimulation (OD) and the levels of this cytokine antemortem TB diagnostic techniques used in goats. In this in serum samples analyzed using the multiplex assay was study we have demonstrated the capacity of dexamethasone poor (rs = 0.215). and ketoprofen recently administered to alter the caprine TB diagnosis by considerably reducing the number of reactors to SIT test. Our results are supported by a previous study of Dexamethasone and ketoprofen Doherty and collaborators in which a significant reduction detection by HPLC in the SFT was observed in TB-infected cattle treated with dexamethasone when compared with untreated control cattle The presence of residues of dexamethasone and ketoprofen (17). The main difference between the aforementioned study in hair and serum samples collected at day 3 was not detected with cattle and our experiment was the administration strategy when employing the HPLC-MS. A minimal amount of residues employed. The parenteral administration of dexamethasone or of this substances were observed in the hair samples of only four ketoprofen 48 h after the PPDs administration demonstrated animals in the dexamethasone group. Nevertheless, the amount the potential use of these substances with fraudulent purposes. detected were far below of the cut-off usually applied for official In this sense, the authorities of different regions in Spain analysis (10 ng/ml), ranging from 0.85 to 1.4 ng/sample. have started to research the possible parenteral administration with fraudulent purposes using different substances such as anti-inflamatory substances in the recent years (19). There is a Discussion high variety of substances and administration protocols which can be applied in ruminants in order to modify the results of the The parenteral administration of dexamethasone or TB diagnostic tests and a lack of knowledge of its effect on the in ketoprofen 48 h after the PPDs inoculation in these experimental vitro techniques. Up to date, there is no evidence to recommend groups significantly reduced the increase in SFT when compared IGRA or diagnostic tests based on the humoral response to to the control group. This reduction, therefore, led to an increase avoid the interferences caused by fraudulent activities on the in false negatives results to the intradermal tests. In this respect, intradermal tests. Recently, IGRA was included as official test several previous studies have evidenced the effect of different for diagnosis of caprine animals in the context of the New anti-inflammatory substances on the immunological response Animal Health Law (Regulation 2016/429) and therefore, it in cattle (17, 18, 27–29). Some of these studies have evaluated would be valuable to evaluate the effect of different fraudulent the effect of dexamethasone on the TB diagnosis techniques in strategies on IGRA results in goats. cattle, such as the intradermal tuberculin test (17) and IGRA The fraudulent activity evaluated in the present study could, (17, 18). Other previous studies have evaluated the in vitro therefore, be directed toward specific animals (those showing effect of dexamethasone, meloxicam or flunixin meglumine on evident reactions at 24–48 h) rather than of all the animals tested. different cell populations in cattle (27–29). However, to the best It is necessary to highlight that in our study, the increase in of the authors’ knowledge, this is the first study to report the the SFT was even lower at the bovine PPD inoculation site Frontiers in Veterinary Science 09 frontiersin.org Ortega et al. 10.3389/fvets.2022.1042428 FIGURE 5 Heatmap of clustering analysis for different cytokines and chemokines analyzed in serum samples collected at day 3 from 10 goats randomly selected from each group (26). when compared to the avian PPD inoculation site, probably as With regard to the effect that the intramuscular inoculation a result of the administration of ketoprofen and dexamethasone of anti-inflammatory substances had on the IGRA results at the same side of the neck as the bovine PPD. According to obtained for animals treated with anti-inflammatory these findings, the dexamethasone or ketoprofen inoculation site drugs, previous studies have shown that the intramuscular affected the SIT test results, since the significant decrease in the administration of dexamethasone reduced the IFN-γ levels SFT was observed only at bovine PPD inoculation site (left- observed in TB-infected cattle (17, 18). Nevertheless, under medial side of the neck in our study). This could, therefore, the conditions of our study, the intramuscular inoculation explain the significant reduction in reactors to the SIT test of dexamethasone or ketoprofen did not lead to a significant in the present study. However, the parenteral administration effect on the number of animals that tested positive to IGRA of dexamethasone or ketoprofen did not lead to a significant since the differences in the number of reactors observed in the reduction in the number of reactors to CIT test, probably owing dexamethasone or ketoprofen groups and the control group to the significant higher SFT at the bovine PPD inoculation site were not significant, probably due to the administration strategy compared to those observed at the avian PPD inoculation site and dose used. The increase in the quantitative values (OD), in the dexamethasone and ketoprofen groups the day of the and subsequently, the number of reactors to IGRA that were intramuscular administration (48 h after the PPDs injection). observed are probably owing to a booster effect associated with This fact and the reduction in the SFT observed at the avian PPD the recent previous PPD administration in the dexamethasone inoculation site in certain animals could explain the lack of a and ketoprofen groups. These results are in agreement with significant reduction in the reactors to CIT test in all groups. a previous study in TB-infected goats that demonstrated that Frontiers in Veterinary Science 10 frontiersin.org Ortega et al. 10.3389/fvets.2022.1042428 a recent previous PPD inoculation had a significant effect of the group: IL-1β, MIP-1β and IL-17A. These results are on IGRA results by increasing the number of reactors when similar to those observed in a previous study in cattle, in which blood was collected 3 days after the intradermal test (16). low concentrations of IL-1β and IL-17A were observed in Nevertheless, this effect was not observed in the control group, interstitial fluid and plasma samples (37). However, these results and is probably associated with the high number of reactors were unexpected, since both cytokines have previously been to IGRA at day 0 in the control group when compared to described as biomarkers of M. bovis infection (38, 39). With dexamethasone group. Another possible reason that could regard to MIP-1β, the low concentration observed is contrary explain this finding might be the posology used: in the study to that of a previous study in cattle in which high plasma levels with cattle, blood was collected from both groups 72 and 36 h were observed (37) and could be associated with differences after dexamethasone (0.2 mg/kg) administration, respectively, between cattle and goats. In our study, IL-8, IP-10 and TNFα whereas in our study, dexamethasone (0.06 mg/kg) was applied were detected at high concentrations in serum samples. In 24 h before blood collection (17). The dose used in our study this respect, previous studies have shown the potential use was considerably lower than that used in cattle, suggesting of IL-8 (40), IP-10 (41–43) and TNFα (39, 44) as biomarkers the possibility that higher doses could have a similar effect of TB-infection in cattle. It is necessary to highlight that the on goats to those observed in cattle (17, 18). With regard low levels of IL-1β, MIP-1β and IL-17A and the high levels to the absence of effect of ketoprofen on IGRA outcome, a of IL-8, IP-10 and TNFα were not associated with a specific previous in vitro study showed demonstrated that non-steroid group and were observed in all apparent M. bovis infected anti-inflammatory substances such as meloxicam did not goats (reactors). The higher concentrations of IL-8, IP-10 and reduce the IFN-γ produced by lymphocytes in cattle, unlike TNFα (proinflammatory response) when compared to IL-4 dexamethasone (29). In this respect, it is necessary to highlight or IL-10 (anti-inflammatory response) suggested a induction that the in vitro study, which showed an evaluation of the of proinflammatory response due to the infection in these effects of dexamethasone and meloxicam on lymphocyte, TB-infected animals, regardless of the study group. With regard employed groups of samples incubated in the absence (controls) to IFN-γ, the poor correlation observed between the IGRA or presence of dexamethasone or meloxicam, and this results (OD) and the levels of this cytokine in serum samples evaluation was performed in vivo in our study (29). Our results was probably associated with the previous PPD stimulation of consequently showed the lack of effect of ketoprofen on the whole blood samples during the performance of IGRA. IGRA results in goats, at least under the conditions of our study. Previous studies have demonstrated that a high variety The differences between the number of positives to P22 of substances and administration protocols can be applied in ELISA in the dexamethasone group and in the control group and ruminants in order to modify the results of the TB diagnostic between the ketoprofen group and the control group were not tests (16–18). This complicates the detection of these fraudulent significant in the present study regardless the cut off employed activities. The development and standardization of tools to (100 or 150 %E), probably owing to the single administration detect these substances is, therefore, essential in order to performed 24 h before these animals were sampled. A recent prevent those activities that affect advances in TB eradication parenteral inoculation of dexamethasone or ketoprofen did not, programmes in ruminants. In this respect, it is important to therefore, have an immunosuppressive effect on the humoral highlight that the administration method affects the detection response. In fact, a significant increase in the Ab levels, expressed of substances used with fraudulent purposes, as shown in a as E%, when using P22 ELISA was detected in the three study previous study (16). Our study demonstrates that certain anti- groups 3 days after the PPD was administered. This booster inflammatory drugs, such as dexamethasone and ketoprofen, effect has been described previously and is used in domestic cannot be detected on hair and serum samples when using ruminants to increase the sensitivity of the skin tests in specific the HPLC technique if they are intramuscularly administered. circumstances (30–35). Although the Ab titres and, therefore, However, the detection of betamethasone topically applied at the sensitivity of the serial use of serological tests are maximized the site of PPD injection using the HPLC technique on hair between 15 or 30 days after the PPD intradermal inoculation, the samples collected from the PPD inoculation site have attained increase in the Ab titres 3 days after the skin test that we describe excellent result previously (16). Using these previous results as in our study has been observed previously in goats (36). a basis, we decided to use hair samples in this study, since The recent parenteral administration of the this sample is easy to collect by using an ante-mortem and dexamethasone and ketoprofen did not significantly affect non-invasive method. Moreover, the time of application was the cytokine/chemokine profile evaluated, since the differences the same in both studies: 48 h after PPD inoculation (16). The in the levels of any cytokine/chemokine observed in serum detection of anti-inflammatory substances, therefore, depends samples obtained from the dexamethasone and ketoprofen on the administration method (topical vs. parenteral), and it groups were not significant with respect to those observed is useful in the case of topical administration. However, the in animals from the control group. Our study showed two significant reduction in the increase in the SFT observed in the cytokines and one chemokine, which were infrequently detected dexamethasone and ketoprofen groups led to a reduction in the and at low concentrations in the serum samples, regardless number of reactors to the intradermal test and, therefore, to Frontiers in Veterinary Science 11 frontiersin.org Ortega et al. 10.3389/fvets.2022.1042428 an increase in false negatives to the intradermal tests. In this Author contributions context, it is necessary to highlight that the lack of detection of these substances does not imply the absence of its effect on the JO and JB wrote the manuscript, performed the literature caprine TB diagnostic technique results, since the reduction in search, and designed the figures. JO, CV, and CN performed the the increase in SFT was observed in animals in whose hair or experiments. JO, LdJ, IS, JG, ÁR, CV, BR, MD, BP, CN, JS-L, JÁ, serum samples the substances were not detected. and JB interpreted the data. All authors reviewed and approved In conclusion, the present study has, for the first time, the manuscript. evaluated the effect of the recent intramuscular administration of dexamethasone and ketoprofen on the results of the SIT and CIT tests, IGRA, and P22 ELISA obtained in goats. Our Funding study demonstrates that corticosteroids and non-steroid anti- inflammatory substances can modify the SIT test results in goats This study was funded by the Analysis of the long-term when they are inoculated 48 h after the PPD administration. caprine tuberculosis eradication process and development of Nevertheless, this recent parenteral administration of the diagnostic tests and control measures for its improvement anti-inflammatory substances did not significantly affect the (GoaTBfree-UCM) Project (PID2019-105155RB-C31). JO was results of the in vitro caprine TB diagnostic techniques. The supported by an FPU contract-fellowship (Formación de detection of these substances by using HPLC on serum or hair Profesorado Universitario) from the Ministerio de Ciencia, samples was challenging, which could be an issue if they were Innovación y Universidades (FPU18/05197). used fraudulently, since the performance of these fraudulent activities are difficult to demonstrate. Additional studies are, therefore, required in order to develop and optimize an Acknowledgments effective protocol to detect these anti-inflammatory substances in different samples. The authors would like to acknowledge Ana Belén Martín (ISCIII), Álvaro Fernández (VISAVET-UCM) and Sergio González (VISAVET-UCM) for their technical assistance. Data availability statement The original contributions presented in the study are Conflict of interest included in the article, further inquiries can be directed to the corresponding author. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics statement The animal study was reviewed and approved by all Publisher’s note handling, testing and sampling procedures were carried out by qualified veterinarians in compliance with European All claims expressed in this article are solely those (86/609/CEE) and Spanish (RD 53/2013) legislation. The of the authors and do not necessarily represent those procedures employed in the current study were similarly of their affiliated organizations, or those of the publisher, approved by an institutional Ethical Committee and ratified the editors and the reviewers. Any product that may be by the local authority (PROEX11/18; Comunidad de Madrid). evaluated in this article, or claim that may be made by Written informed consent was obtained from the owners for the its manufacturer, is not guaranteed or endorsed by the participation of their animals in this study. publisher. References 1. Rodríguez S, Bezos J, Romero B, De Juan L, Álvarez J, Castellanos E, et al. 3. Malone KM, Gordon SV. Mycobacterium tuberculosis complex members Mycobacterium caprae infection in livestock and wildlife, Spain. Emerg Infect Dis. adapted to wild and domestic animals. AdvExp Med Biol. (2017) 1019:135– (2011) 17:532–5. doi: 10.3201/eid1703.100618 54. doi: 10.1007/978-3-319-64371-7_7 2. Gortázar C, CheAmat A, O’Brien DJ. Open questions and recent advances 4. Montali RJ, Mikota SK, Cheng LI. Mycobacterium tuberculosis in zoo in the control of a multi-host infectious disease: animal tuberculosis. Mamm Rev. and wildlife species. Rev Sci Tech. (2001) 20:291–303. doi: 10.20506/rst.20.1. (2015) 45:160–75. doi: 10.1111/mam.12042 1268 Frontiers in Veterinary Science 12 frontiersin.org Ortega et al. 10.3389/fvets.2022.1042428 5. Pesciaroli M, Alvarez J, Boniotti MB, Cagiola M, Di Marco V, Marianelli C, 26. Babicki S, Arndt D, Marcu A, Liang Y, Grant JR, Maciejewski A, et et al. Tuberculosis in domestic animal species. Res Vet Sci. (2014) (97 Suppl):S78– al. Heatmapper: web-enabled heat mapping for all. Nucleic Acids Res. (2016). 85. doi: 10.1016/j.rvsc.2014.05.015 doi: 10.1093/nar/gkw419. [Epub ahead of print]. 6. Bezos J, Alvarez J, Romero B, Aranaz A, Juan L. Tuberculosis in goats: 27. Maślanka T, Jaroszewski JJ, Markiewicz W, Jakubowski P. Evaluation of the assessment of current in vivo cell-mediated and antibody-based diagnostic assays. influence of meloxicam and flunixinmeglumine on the apoptosis of peripheral Vet J. (2012) 191:161–5. doi: 10.1016/j.tvjl.2011.02.010 blood CD4+ and CD8+ T cells in calves. Pol J Vet Sci. (2010) 13:3–12. 7. Collins JD. Tuberculosis in cattle: strategic planning for the future. Vet 28. Maslanka T, Jaroszewski JJ. In vitro effects of dexamethasone on Microbiol. (2006) 112:369–81. doi: 10.1016/j.vetmic.2005.11.041 bovine CD25+CD4+ and CD25-CD4+ cells. Res Vet Sci. (2012) 93:1367– 79. doi: 10.1016/j.rvsc.2012.01.018 8. Waters WR, Palmer MV, Buddle BM, Vordermeier HM. Bovine tuberculosis vaccine research: historical perspectives and recent advances. Vaccine. (2012) 29. Maslanka T, Jaroszewski JJ, Markiewicz W, Jasiecka A, Ziolkowski H, 30:2611–22. doi: 10.1016/j.vaccine.2012.02.018 Jedrzkiewicz D. Effects of dexamethasone and meloxicam on bovine CD25+ CD8+ and CD25- CD8+ T cells–in vitro study. Res Vet Sci. (2013) 94:662– 9. Eurostat, 2021. (Statistical Office of the European Union). (2021). Available 74. doi: 10.1016/j.rvsc.2012.12.005 online at: https://ec.europa.eu/eurostat/web/products-data-sets/-/apro_mt_lsgoat (accessed July 14, 2022). 30. Bezos J, Roy A, Infantes-Lorenzo JA, González I, Venteo A, Romero B, et al. The use of serological tests in combination with the intradermal 10. Roy A, Infantes-Lorenzo JA, de la Cruz ML, Dominguez L, tuberculin test maximizes the detection of tuberculosis infected goats. Vet Alvarez J, Bezos J. Accuracy of tuberculosis diagnostic tests in small ImmunolImmunopathol. (2018) 199:43–52. doi: 10.1016/j.vetimm.2018.03.006 ruminants: a systematic review and meta-analysis. PrevVetMed. (2020) 182:105102. doi: 10.1016/j.prevetmed.2020.105102 31. Buddle BM, Wilson T, Luo D, Voges H, Linscott R, Martel E, et al. Evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of bovine 11. Bezos J, Alvarez J, Juan L, Romero B, Rodriguez S, Castellanos E, et al. Factors tuberculosis from milk samples from dairy cows. Clin Vaccine Immunol. (2013) influencing the performance of an interferon-gamma assay for the diagnosis of 20:1812–6. doi: 10.1128/CVI.00538-13 tuberculosis in goats. Vet J. (2011) 190:131–5. doi: 10.1016/j.tvjl.2010.09.026 32. Casal C, Diez-Guerrier A, Alvarez J, Rodriguez-Campos S, Mateos 12. Good M, Bakker D, Duignan A, Collins DM. The history of in vivo A, Linscott R, et al. Strategic use of serology for the diagnosis of bovine tuberculin testing in bovines: tuberculosis, a “one health” issue. Front Vet Sci. tuberculosis after intradermal skin testing. Vet Microbiol. (2014) 170:342– (2018) 5:59. doi: 10.3389/fvets.2018.00059 51. doi: 10.1016/j.vetmic.2014.02.036 13. Schiller I, Vordermeier HM, Waters WR, Whelan AO, Coad M, 33. Casal C, Infantes JA, Risalde MA, Díez-Guerrier A, Domínguez M, Moreno Gormley E, et al. Bovine tuberculosis: effect of the tuberculin skin test on in I, et al. Antibody detection tests improve the sensitivity of tuberculosis diagnosis in vitro interferon gamma responses. Vet ImmunolImmunopathol. (2010) 136:1– cattle. Res Vet Sci. (2017) 112:214–21. doi: 10.1016/j.rvsc.2017.05.012 11. doi: 10.1016/j.vetimm.2010.02.007 34. Gutiérrez M, Tellechea J, García Marín JF. Evaluation of cellular and 14. Good M, Clegg TA, Costello E, More SJ. The comparative performance of the serological diagnostic tests for the detection of Mycobacterium bovis-infected goats. single intradermal test and the single intradermal comparative tuberculin test in Vet Microbiol. (1998) 62:281–90. doi: 10.1016/S0378-1135(98)00217-X Irish cattle, using tuberculin PPD combinations of differing potencies. Vet J. (2011) 190:e60–e5. doi: 10.1016/j.tvjl.2011.01.005 35. Waters WR, Palmer MV, Stafne MR, Bass KE, Maggioli MF, Thacker TC, et al. Effects of serial skin testing with purified protein derivative on the level and quality 15. Ghielmetti G, Landolt P, Friedel U, Morach M, Hartnack S, Stephan R, et al. of antibodies to complex and defined antigens in Mycobacterium bovis-infected Evaluation of three commercial interferon-gamma assays in a bovine tuberculosis cattle. Clin Vaccine Immunol. (2015) 22:641–9. doi: 10.1128/CVI.00119-15 free population. Front Vet Sci. (2021) 8:682466. doi: 10.3389/fvets.2021.682466 36. O’Brien A, Whelan C, Clarke JB, Hayton A, Watt NJ, Harkiss GD. 16. Ortega J, Roy A, Díaz-Castillo A, de Juan L, Romero B, Sáez-Llorente Serological Analysis of Tuberculosis in Goats by Use of the Enferplex Caprine TB JL, et al. Effect of the topical administration of corticosteroids and tuberculin Multiplex Test. Clin Vaccine Immunol. (2017) 24:e00518–16. doi: 10.1128/CVI.00 pre-sensitisation on the diagnosis of tuberculosis in goats. BMC Vet Res. (2022) 518-16 18:58. doi: 10.1186/s12917-022-03156-0 37. Steinbach S, Jalili-Firoozinezhad S, Srinivasan S, Melo MB, Middleton S, 17. Doherty ML, Bassett HF, Quinn PJ, Davis WC, Monaghan ML. Effects Konold T, et al. Temporal dynamics of intradermal cytokine response to tuberculin of dexamethasone on cell-mediated immune responses in cattle sensitized to in Mycobacterium bovis BCG-vaccinated cattle using sampling microneedles. Sci Mycobacterium bovis. Am J Vet Res. (1995) 56:1300–6. Rep. (2021) 11:7074. doi: 10.1038/s41598-021-86398-6 18. Goff BS. Effect of dexamethasone treatment of tuberculous cattle on results of 38. Aranday-Cortes E, Bull NC, Villarreal-Ramos B, Gough J, Hicks D, Ortiz- the gamma-interferon test for Mycobacterium bovis. Vet ImmunolImmunopathol. Pelaez A, et al. Upregulation of IL-17A, CXCL9 and CXCL10 in early-stage (1996) 53:39–47. doi: 10.1016/0165-2427(96)05601-2 granulomas induced by Mycobacterium bovis in cattle. Transbound Emerg Dis. 19. Regal P, Lamas A, Díaz-Bao M, Ortega J, Bezos J, Cepeda A, et al. Bovine (2013) 60:525–37. doi: 10.1111/j.1865-1682.2012.01370.x tuberculosis in Spain: emerging residue perspectives. Euro Residue IX Conference 39. Jones GJ, Pirson C, Hewinson RG, Vordermeier HM. Simultaneous on Residues of Veterinary Drugs in Food. St. Michielsgestel, The Netherlands. measurement of antigen-stimulated interleukin-1 beta and gamma (2022). p. 23–25. Available online at: https://www.euroresidue.eu/posters-2022/ interferon production enhances test sensitivity for the detection of various-subjects Mycobacterium bovis infection in cattle. Clin Vaccine Immunol. (2010) 20. Ortega J, Roy Á, Álvarez J, Sánchez-Cesteros J, Romero B, Infantes- 17:1946–51. doi: 10.1128/CVI.00377-10 Lorenzo JA, et al. Effect of the inoculation site of bovine and avian Purified 40. Gao X, Guo X, Li M, Jia H, Lin W, Fang L, et al. Interleukin 8 and Pentaxin Protein Derivatives (PPDs) on the performance of the intradermal tuberculin (C-Reactive Protein) as potential new biomarkers of bovine tuberculosis. J Clin test in goats from tuberculosis-free and infected herds. Front Vet Sci. (2021) Microbiol. (2019) 57:e00274–19. doi: 10.1128/JCM.00274-19 8:722825. doi: 10.3389/fvets.2021.722825 41. Bernitz N, Kerr TJ, Goosen WJ, Higgitt RL, de Waal C, Clarke C, 21. Roy A, Tome I, Romero B, Lorente-Leal V, Infantes-Lorenzo JA, Domínguez et al. Impact of Mycobacterium bovis-induced pathology on interpretation of M, et al. Evaluation of the immunogenicity and efficacy of BCG and MTBVAC QuantiFERON(R)-TB Gold assay results in African buffaloes (Syncerus caffer). vaccines using a natural transmission model of tuberculosis. Vet Res. (2019) Vet Immunol Immunopathol. (2019) 217:109923. doi: 10.1016/j.vetimm.2019.10 50:82. doi: 10.1186/s13567-019-0702-7 9923 22. Infantes-Lorenzo JA, Moreno I, Risalde MA, Roy A, Villar M, Romero B, 42. Palmer MV, Thacker TC, Rabideau MM, Jones GJ, Kanipe C, Vordermeier et al. Proteomic characterisation of bovine and avian purified protein derivatives HM, et al. Biomarkers of cell-mediated immunity to bovine tuberculosis. and identification of specific antigens for serodiagnosis of bovine tuberculosis. Clin Vet Immunol Immunopathol. (2020) 220:109988. doi: 10.1016/j.vetimm.2019.10 Proteomics. (2017) 14:36. doi: 10.1186/s12014-017-9171-z 9988 23. Infantes-Lorenzo JA, Moreno I, Roy A, Risalde MA, Balseiro A, de Juan L, 43. Parsons SD, McGill K, Doyle MB, Goosen WJ, van Helden PD, et al. Specificity of serological test for detection of tuberculosis in cattle, goats, Gormley E. Antigen-specific IP-10 release is a sensitive biomarker sheep and pigs under different epidemiological situations. BMC Vet Res. (2019) of Mycobacterium bovis infection in cattle. PLoS ONE. (2016) 15:70. doi: 10.1186/s12917-019-1814-z 11:e0155440. doi: 10.1371/journal.pone.0155440 24. R Development Core Team. An Official Part of the Free Software Foundation’s 44. Waters WR, Palmer MV, Whipple DL, Carlson MP, Nonnecke BJ. Diagnostic GNU Project. (2021). Available online at: https://www.R-project.org/ (accessed implications of antigen-induced gamma interferon, nitric oxide, and tumor September 8, 2022). necrosis factor alpha production by peripheral blood mononuclear cells from 25. Abramson JH, WINPEPI. (PEPI-for-Windows): computer programs for Mycobacterium bovis-infected cattle. Clin Diagn Lab Immunol. (2003) 10:960– epidemiologists. Epidemiol Perspect Innov. (2004) 1:6. doi: 10.1186/1742-5573-1-6 6. doi: 10.1128/CDLI.10.5.960-966.2003 Frontiers in Veterinary Science 13 frontiersin.org