TYPE Original Research PUBLISHED 11 August 2023 DOI 10.3389/fmicb.2023.1236834 Comparison of the pathological OPEN ACCESS outcome and disease progression of two Mycobacterium caprae EDITED BY Hong Yin, Chinese Academy of Agricultural Sciences, China REVIEWED BY experimental challenge models in Heike Uta Köhler, Friedrich-Loeffler-Institut, Germany Elisabeth M. Liebler-Tenorio, goats: endobronchial inoculation Friedrich-Loeffler-Institut, Germany *CORRESPONDENCE vs. intranasal nebulization Bernat Pérez de Val bernat.perez@irta.cat Enric Vidal Cristian Melgarejo1,2 , Alex Cobos1,2,3 , Carles Planas4 , enric.vidal@irta.cat Jaume Fondevila2 , Maite Martín2 , Zoraida Cervera2 , † These authors have contributed equally to this work and share last authorship Guillermo Cantero1,2 , Xavier Moll4,5 , Yvonne Espada4,5 , RECEIVED 08 June 2023 Mariano Domingo1,2,3 , Enric Vidal1,2*† and Bernat Pérez de Val1,2*† ACCEPTED 25 July 2023 1 Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal, Campus PUBLISHED 11 August 2023 de la Universitat Autònoma de Barcelona, Bellaterra, Spain, 2 IRTA, Programa de Sanitat Animal, Centre de CITATION Recerca en Sanitat Animal, Campus de la Universitat Autònoma de Barcelona, Bellaterra, Spain, 3 Melgarejo C, Cobos A, Planas C, Fondevila J, Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Bellaterra, Spain, 4 Martín M, Cervera Z, Cantero G, Moll X, Fundació Hospital Clínic Veterinari, Universitat Autònoma de Barcelona, Bellaterra, Spain, 5 Departament Espada Y, Domingo M, Vidal E and Pérez de de Medicina y Cirugía Animals, Universitat Autònoma de Barcelona, Bellaterra, Spain Val B (2023) Comparison of the pathological outcome and disease progression of two Mycobacterium caprae experimental challenge models in goats: endobronchial inoculation vs. Background: Goats are natural hosts of tuberculosis (TB) and are a valid animal intranasal nebulization. model to test new vaccines and treatments to control this disease. In this study, Front. Microbiol. 14:1236834. a new experimental model of TB in goats based on the intranasal nebulization of doi: 10.3389/fmicb.2023.1236834 Mycobacterium caprae was assessed in comparison with the endobronchial route COPYRIGHT of infection. © 2023 Melgarejo, Cobos, Planas, Fondevila, Martín, Cervera, Cantero, Moll, Espada, Methods: Fourteen animals were divided into two groups of seven and challenged Domingo, Vidal and Pérez de Val. This is an through the endobronchial (EB) and intranasal (IN) routes, respectively. Clinical open-access article distributed under the terms of the Creative Commons Attribution License signs, rectal temperature, body weight, and immunological responses from (CC BY). The use, distribution or reproduction blood samples were followed up throughout the experiment. All goats were in other forums is permitted, provided the euthanized at 9 weeks post-challenge. Gross pathological examination, analysis original author(s) and the copyright owner(s) are credited and that the original publication in of lung lesions using computed tomography, and bacterial load quantification in this journal is cited, in accordance with pulmonary lymph nodes (LNs) by qPCR were carried out. accepted academic practice. No use, distribution or reproduction is permitted which Results: The IN-challenged group showed a slower progression of the infection: does not comply with these terms. delayed clinical signs (body weight gain reduction, peak of temperature, and apparition of other TB signs) and delayed immunological responses (IFN-γ peak response and seroconversion). At the end of the experiment, the IN group also showed significantly lower severity and dissemination of lung lesions, lower mycobacterial DNA load and volume of lesions in pulmonary LN, and higher involvement of the nasopharyngeal cavity and volume of the lesions in the retropharyngeal LN. Conclusion: The results indicated that the IN challenge with M. caprae induced pathological features of natural TB in the lungs, respiratory LN, and extrapulmonary organs but extremely exaggerating the nasopharyngeal TB pathological features. On the other hand, the EB route oversized and accelerated the pulmonary TB lesion progression. Our results highlight the need to refine the inoculation routes in the interest of faithfully reproducing the natural TB infection when evaluating new vaccines or treatments against the disease. KEYWORDS tuberculosis, Mycobacterium caprae, goat, experimental infection, intranasal, endobronchial Frontiers in Microbiology 01 frontiersin.org Melgarejo et al. 10.3389/fmicb.2023.1236834 1. Introduction of goats with M. caprae by intranasal nebulization in comparison with the endobronchial route. The rationale was to use a more Tuberculosis (TB) is an infectious disease caused by natural access path of mycobacteria to the respiratory tract than the mycobacteria belonging to the Mycobacterium tuberculosis endobronchial route to reproduce more precisely the spectrum of complex (MTBC) that affects humans and a wide range of lesions of goat TB found in field cases. domestic animals and wildlife. TB in domestic goats is mainly caused by Mycobacterium caprae, a member of the MTBC (Aranaz et al., 2003). The disease has an economic impact resulting from 2. Materials and methods production losses (Seva et al., 2002; Daniel et al., 2009), and infected goats pose a risk of infection to other animal species 2.1. Animals and experimental infections (Napp et al., 2013; Cano-Terriza et al., 2018; Vidal et al., 2018) and humans (Rodríguez et al., 2011). Fourteen 3–4 months old female Pyrenean goats, from an TB in goats is usually transmitted through the aerogenous officially tuberculosis-free herd, located in the Catalan Pyrenees route, and it is common to find lesions mainly in the respiratory (Spain), were chosen for this study. Experimental animals were tract (Domingo et al., 2014). Goats develop caseous-necrotizing transferred to the IRTA-CReSA Biosafety Level 3 (BSL-3) facility cavitary lesions similar to those observed in human TB (Pérez de (Catalonia, Spain), where they were weighted and randomly Val et al., 2011; Sanchez et al., 2011), whereas these lesions are divided into two experimental groups of seven animals each, rarely developed in cattle (Buddle et al., 2016). Different approaches challenged by the endobronchial (EB) and intranasal (IN) routes, exist to control livestock TB, and the test and slaughter strategy is respectively. According to body weights, corrections to ensure the most widely implemented, particularly in cattle. Vaccination homogenous distribution between the groups were applied, and against TB could be considered as an alternative approach. then, the animals were housed in two experimental boxes with However, cattle vaccination against TB is currently forbidden in animals 4–3 and 3–4 from the EB and IN groups in each box, the EU (Council Directive 78/52/EEC) due to interference with respectively. The animals were fed hay, alfalfa, feed, and mineral currently used TB diagnostic tools. The goat model of TB has salt and had access to water ad libitum throughout the experiment. proven to be a suitable one to study new TB vaccines for ruminants After 1 week of acclimatization, the animals were sedated and humans and for a better understanding of the pathogenesis of intramuscularly with acepromazine maleate (0.05 mg/kg) and the disease (Ramirez et al., 2003; Pérez de Val et al., 2011, 2013; butorphanol (0.2 mg/kg) and subsequently anesthetized with Wedlich et al., 2022). intravenous administration of propofol (5 mg/kg) and midazolam In the different TB studies using the goat model, the infection (0.2 mg/kg). Afterward, the animals were challenged with 0.5 ml of has typically been carried out through the endobronchial route a M. caprae field strain (Balseiro et al., 2017) at a suspension of 2 (Pérez de Val et al., 2011, 2012, 2013; Arrieta-Villegas et al., × 103 CFU/ml. Animals of the EB group were challenged through 2018, 2020; Melgarejo et al., 2022), intratracheal aerosolization the endobronchial route as previously described (Pérez de Val et al., (Gonzalez-Juarrero et al., 2013), and transthoracic injection (Bezos 2011). et al., 2010, 2015), all of them inducing primarily pulmonary TB The animals of the IN group were challenged by using a syringe- lesions. In particular, the endobronchial approach allows for an adapted device for atomized spraying (MADgic R , Wolfe Tory accelerated progression of pulmonary TB, which is positive in Medical, Inc., Salt Lake City, USA). This device consists of a flexible terms of reducing the duration of the experiments. However, if cannula measuring 21.6 cm in length and 4.82 mm in width with a the infectious dose is not well-adjusted, exaggeratedly large lung nebulizer located at its end that generates droplets of 30–100 µm lesions may be produced. These large lesions do not only differ in diameter, according to the manufacturer’s instructions. The from the common natural presentation of the disease but also cannula was inserted into the nasal cavity ∼10–15 cm depending might preclude a proper evaluation of the vaccine effect (Arrieta- on the size of the animal. Each side of the nasal cavity received Villegas et al., 2020). A completely different experimental approach 0.5 ml of the same M. caprae strain at a suspension of 103 CFU/ml is the “natural” infection by long-term direct-contact exposure of without anesthesia. experimental goats with infected animals in controlled conditions After the challenge, all animals were daily monitored for clinical (Bezos et al., 2017; Roy et al., 2018, 2019). The advantage of signs by a veterinarian to assess whether any of the animals reached this approach is that it induces all the natural features of the the endpoint criteria. The clinical evaluation included 0–3 scoring infection, but other aspects such as infection dose or infection of each of the following five parameters: body condition, mental time point remain uncontrolled, and it dramatically increases state/behavior, weight loss, fever, and the presence of respiratory the duration of the experiment. Nowadays, in vitro/ex vivo and signs (0: absence of respiratory signs; 1: mild dyspnea and/or in silico experimental models are being developed to replace or occasional cough; 2: moderate dyspnea and continued coughing; reduce animal models in preclinical studies of vaccine candidates and 3: marked dyspnea, continued coughing, and/or severe nasal against TB (Tanner et al., 2016; Català et al., 2020). However, the discharge). Any global score higher than 11 and/or the presence of optimization of these approaches requires a comparison to well- qualitative criteria such as prostration, lack of movement, or severe characterized animal models that can be used as “gold standard”. respiratory distress resulted in euthanasia. Rectal temperature Altogether, an accurate standardization of a goat model of TB is and body weight were measured each week. Heparinized blood critical to predicting the efficacy of new vaccine candidates. This samples were collected at weeks 0 (before challenge), 3, 5, 7, and study aimed to evaluate and characterize the experimental infection 9 (endpoint) for immunological assays. Frontiers in Microbiology 02 frontiersin.org Melgarejo et al. 10.3389/fmicb.2023.1236834 2.2. Ethics statement was between 110 and 150%, and positive (seroconversion) when E% ≥ 150%. All the procedures with experimental animals carried out during the study were approved by the Animal Welfare Committee of the Generalitat de Catalunya (Project Ref. #10794), in 2.6. Necropsy, pathological examination, accordance with the European Union legislation for the protection and computed tomography of experimental animals (86/609/CEE, 91/628/EEC, 92/65/EEC, and 90/425/EEC). At week 9 post-challenge, all goats were sedated intramuscularly by an injection of acepromazine maleate (0.1 mg/kg) and butorphanol (0.2 mg/kg) and subsequently euthanized 2.3. Antigens and reagents with an intravenous overdose of sodium pentobarbital. At necropsy, the size of TB-compatible lesions in the retropharyngeal M. bovis tuberculin (PPD-B, 2,500 IU/ml) was obtained from (right and left), mediastinal (cranial and caudal), and CZ vaccines (Porriño, Spain). The specific recombinant antigens of tracheobronchial (LN) lymph nodes was measured, and the MTBC ESAT-6, CFP-10, and MPB83 were obtained from Lionex volume of lesions was inferred using the volume formula of the (Braunschweig, Germany) at a concentration of 500 µg/ml each. most similar 3D-geometrical morphology (including sphere, ESAT-6 and CFP-10 were mixed 1:1 in an antigenic (E/C) cocktail. cylinder, or prism), as previously described (Balseiro et al., The P22 antigenic complex was produced by immunopurification 2017). Other visible lesions in extrapulmonary tissues were also of PPD-B (CZ vaccines) as described previously (Infantes-Lorenzo recorded and fixed in 10% buffered formalin to be confirmed by et al., 2017) and was supplied by the Carlos III Research Institute histopathology and Ziehl–Neelsen staining to detect acid-resistant (Madrid, Spain) at a concentration of 500 µg/ml. bacilli. The lungs were fixed by airway instillation followed by immersion in formalin, as previously described (Pérez de Val et al., 2011), and then scanned by computed tomography (CT) 2.4. Whole blood IFN-γ release assay using a 16-slice multidetector scanner (Brivo CT-385, General Electric Healthcare, Madrid, Spain). CT analysis was performed Approximately 10 ml of whole blood was collected at as previously described (Balseiro et al., 2017). In brief, total the abovementioned time points from the jugular vein using volumes of the lungs and TB lesions were measured using heparinized tubes. Blood samples were maintained at room volume representation images with different density patterns (i.e., temperature for <1 h when they were stimulated in 96-well cell solid, mineralized, and cavitary lesions), and the total volume of culture plates (Eppendorf, Hamburg, Germany) with PPD-B, P22 mineralization was calculated using 100–300 Hounsfield units. (except at week 7), and E/C at a final concentration of 20 µg/ml each. Phosphate-buffered saline was used as an unstimulated control. The samples were incubated at 37◦ C and 5% CO2 for 18 2.7. Quantification of bacterial DNA ± 2 h. Plasma supernatants were collected after centrifugation at 18 g for 10 min, and the released IFN-γ was measured by ELISA After macroscopic pathological evaluation, mediastinal and (ID Screen R Ruminant IFN-g kit, ID.vet, Grabels, France). Optical tracheobronchial LNs were collected and stored at −20◦ C until densities of the ELISA plates were read at 450 nm (OD450nm ) using they were processed for bacteriology. The whole LNs were then a spectrophotometer (Biotek Power Wave XS R , Agilent, Santa thawed and homogenized in 10 ml of sterile-distilled water using Clara, USA). Antigen-specific IFN-γ responses were calculated as a homogenizer (Masticator, IUL Instruments, Barcelona, Spain). OD450nm of the antigen-stimulated well minus OD450nm of the An aliquot of 1 ml of each homogenate was inactivated at 75◦ C unstimulated well (1OD450 nm ). for 1 h for bacterial DNA quantification. In parallel, an aliquot of 100 ml of the master seed of M. caprae (∼108 CFU/ml), used for the challenged inoculum, was inactivated and then diluted in eight 10- 2.5. Antibody detection assays fold dilutions to establish the standard curve. DNA samples were extracted using an ID GeneTM spin universal extraction kit (ID.vet) Plasma samples from all experimental animals were analyzed and amplified with the Mycobacterium tuberculosis complex in duplicate to follow antibody responses against MTBC after the duplex kit (ID.vet), following the manufacturer’s instructions. challenge. Indirect ELISAs were used to detect total IgG against The amplification was performed in a 7500 fast real-time PCR MPB83 antigen and P22 antigenic complex, respectively. The two system (Applied Biosystems, Waltham, MA, USA). The genomic ELISAs were performed as previously described (Pérez de Val equivalents of CFU were calculated as previously described (Vidal et al., 2017; Infantes-Lorenzo et al., 2019). MPB83-IgG levels were et al., 2022). calculated as OD450nm of the antigen-coated well minus OD450nm of the uncoated well (1OD450nm ). A sample was considered positive when 1OD450nm ≥ 0.05. P22-IgG levels were calculated as a 2.8. Data analysis percentage of ELISA (E%) = [mean OD450nm of the antigen-coated well/(2 × mean negative control OD450nm )] × 100. A sample was A completely randomized design was performed to compare considered negative when E% ≤ 100%, non-conclusive when E% the effects of M. caprae infection (primary factor) by the EB and Frontiers in Microbiology 03 frontiersin.org Melgarejo et al. 10.3389/fmicb.2023.1236834 IN routes. First, all data distributions were analyzed by using significant at week 5 (P < 0.05, Figure 3B), although a stagnation the Shapiro–Wilk normality test. Temperature and body weight in body weight increase was observed from week 5 onward. In were compared using a unidirectional unpaired t-test. Antigen- contrast, animals belonging to the EB group showed a mild increase specific immune responses (measured by IGRA, IgG-ELISA), lesion in mean body weight from week 6 to the end of the experiment, volumes, and mycobacterial DNA load were compared using the where the difference in mean cumulative weight gain (1Kg, Kg at non-parametric Mann–Whitney test. GraphPad Prism version 8.0.0 weeks 9–0) between the IN and EB groups was minimal (4.2 ± 3.3 (San Diego, CA, USA) was used for statistical analyses. 95% CI and 3.5 ± 2.9 95%CI, respectively). The presence or absence of clinical signs, other than weight loss or fever, compatible with TB was recorded in all challenged 3. Results animals until the end point of the experiment (Figure 3C). The observed signs included cough, dyspnea, eye discharge, enlarged 3.1. Endobronchial challenge induced lymph nodes, runny nose, and anorexia. An animal of the EB group earlier and stronger immune responses showed clinical signs at week 1, 4 at week 2, and 6 from week 3 to the endpoint, whereas a 2-week delay in the onset of clinical signs Mean antigen-specific IFN-γ responses in whole blood before was observed in the IN group: 1, 4, and 6 animals at weeks 3, 4, and and after the challenge for each treatment group are presented in from 5 to the endpoint, respectively (Figure 3C). The proportion of Figure 1. An increase in IFN-γ-specific responses to PPD-B, P22, animals with clinical signs was significantly higher in the EB group and E/C antigens was detected in both groups after the challenge at weeks 2 (P < 0.01), 3 (P < 0.01), and 4 (P < 0.05) compared (week 0, Figures 1A–C). Animals of the EB group showed a peak- with the IN group. All experimental animals showed clinical signs up response of IFN-γ to the three antigens at weeks 3 and 5, which at weeks 8 and 9. was significantly higher than in animals of the IN group (P < 0.01; except P < 0.05 for PPD-B at week 5). IFN-γ-specific responses in intranasally challenged animals increased progressively over weeks, while IFN-γ levels elicited after the endobronchial challenge 3.3. Endobronchially challenged animals decreased at the end of the experiment when responses in both showed more severe lung lesions groups tended to converge (no significant differences at weeks 7 and 9). All goats showed TB lesions at necropsy. Lung lesions were IgG levels to the MPB83 antigen and the P22 antigenic complex, quantitatively assessed by CT. All animals challenged by the EB as well as individual seroconversion, were studied using two ELISAs route (7/7) had lung lesions, while only 4/7 animals of the IN group throughout the study (Figure 2). Endobronchial challenge induced presented lung lesions, and the severity and dissemination of these an early increase in MPB83 IgG-specific responses (a peak-up lesions were very much lower compared with those found in the response at weeks 5 and 7), and all EB animals seroconverted at EB group. Figure 4 shows the extension of lung TB granulomas week 5 p.c. On the contrary, only a mild response was detected in IN expressed as the volume of lung lesions, the intrapulmonary spread animals, seroconversion was not detected in 2 of them throughout of TB lesions expressed as the number of affected lung lobes, and the experiment, and the other five animals seroconverted between the degree of development of lung TB lesions expressed as the weeks 5 and 9 (Figure 2A). In comparison with MPB83, IgG volume ratio between mineralization and lesion of each group. The responses to P22 were more progressive in the EB group, reaching EB group showed a significantly larger median volume of lung a peak at the end of the experiment (week 9), but seroconversion lesions compared with the IN group (P < 0.001, Figure 4A). In was detected in six of seven animals. IgG responses to P22 addition, the EB group showed a higher number of affected lobes were practically undetectable in the IN group throughout the compared with the IN group (P < 0.01, Figure 4B). Five out of experiment, and only one animal showed a clear seroconversion seven EB animals showed TB lesions in all seven pulmonary lobes, (Figure 2B). while only one IN animal showed lesions in six pulmonary lobes. The EB group also showed a significantly greater median volume of lung mineralization (18.36 cm3 , 95% CI: 2. 634–84.53, P < 0.001) 3.2. Disease-related clinical signs appeared compared with the IN group (0 cm3 , 95% CI: 0–0.72), as well as higher median proportion of mineralization of lung lesions (P < later in intranasally challenged goats 0.01; Figure 4C) indicative of a faster progression of pulmonary TB infection. Rectal temperature, body weight, and clinical signs of TB were weekly recorded throughout the experiment (Figure 3). In the EB group, a peak of mean rectal temperature was observed between weeks 2 and 4 p.c., being statistically significantly 3.4. Intranasal challenge caused more higher compared with the IN group (P < 0.01, weeks 2 and 4; P < severe lesions in the head but less 0.05, week 3; Figure 3A). A mild peak was also observed in the IN generalized TB forms group between weeks 5 and 7, although still remained slightly lower than the EB group, and decreased at week 8 to be significantly lower All intranasally challenged animals showed lesions in than the EB group again (P < 0.05). retropharyngeal (RF) LNs, while these lesions were only detected The mean body weight gain was greater in the IN group in 2/7 animals of the EB group, and the median volume of lesions compared with the EB group since week 4, being statistically in the RF LNs was significantly higher in the IN group compared Frontiers in Microbiology 04 frontiersin.org Melgarejo et al. 10.3389/fmicb.2023.1236834 FIGURE 1 Whole blood IFN-γ responses before and after challenge: the graph shows the IFN-γ levels measured by ELISA. The results are expressed as 1OD450mn ± 95% CI. (A) Response against bovine tuberculin (PPD-B). (B) Response to the P22 complex. (C) Response against E/C antigen cocktail. *P < 0.05, **P < 0.01, Mann–Whitney test. Groups: Endobronchial (n = 7, green) and Intranasal (n = 7, orange). Two animals of the endobronchial group were humanely sacrificed at week 7 and one animal of the group Intranasal. FIGURE 2 Antibody responses before and after the challenge. The figure shows the mean levels of IgG against MPB83 and P22 antigens measured by ELISA throughout the study. (A) MPB83-ELISA results are expressed as 1OD450mn ± 95% CI. A sample was considered positive when 1OD450nm ≥ 0.05. (B) P22-ELISA results are expressed as E%. E% = [mean OD450nm of the antigen-coated well/(2 × mean negative control OD450nm)] × 100. A sample is considered negative when it is ≤100%, inconclusive between 110 and 150% and seroconverted when the percentage is ≥150%. The dashed horizontal line shows the cutoff point for positivity. *P < 0.05, **P < 0.01, ***P < 0.001. Mann–Whitney test. The table indicates the numbers of animals and their seroconversion status: negative/inconclusive/positive. Groups: endobronchial (EB, N = 7, a N = 5 at W9) and intranasal (IN, N = 7, b N = 6 at W9). with the EB group (P < 0.01, Figure 5C). In contrast, only 3/7 All extrapulmonary lesions macroscopically compatible with animals that received the IN challenge developed TB lesions in TB were confirmed as such by histopathological examination. In pulmonary LNs, whereas these lesions were macroscopically found general, the IN group presented major affectation of the head, in all EB-challenged ones. The median volume of TB lesions in whereas more severe TB-compatible lesions in the thoracic and pulmonary LN was significantly higher in the EB group compared abdominal cavities were found in the EB group. Four out of with the IN group (P < 0.001, Figure 5A), and the bacterial seven animals of the IN group had an intense granulomatous DNA load in pulmonary LNs, estimated as CFU equivalents by inflammatory infiltrate in the nasal cavity (see Figures 6E, F), qPCR, was also significantly higher in the EB group (P < 0.05, and the three animals had lesions in the retropharyngeal and/or Figure 5B). submandibular lymph nodes. Frontiers in Microbiology 05 frontiersin.org Melgarejo et al. 10.3389/fmicb.2023.1236834 FIGURE 3 Rectal temperature, body weight, and clinical signs post-challenge. (A) The weekly mean rectal temperature of each group expressed in ◦ C ± 95% CI. (B) Weekly mean cumulative body weight increases of each group expressed in 1Kg (Kg at each week post-challenge minus Kg at week 0) ± 95% CI. (C) The percentage of animals with the presence of TB-related clinical signs in each group. The clinical signs observed were cough, dyspnea, eye discharge, enlarged lymph nodes, nasal discharge, and anorexia (the groups were compared using the chi-square test). Groups: endobronchial (green, N = 7; N = 5 at weeks 8 and 9) intranasal (orange, N = 7; N = 6 at weeks 8 and 9). Body temperature and weight were compared using the one-way unpaired t-test. *P < 0.05, **P < 0.01. FIGURE 4 Lung pathological findings in endobronchially (EB) and intranasally (IN) M. caprae-challenged goats. (A) Volumes of lung TB lesions expressed in cm3 measured by computed tomography (CT) scan. (B) Number of lung lobes with TB lesions. (C) The ratio between the volume of mineralization and the volume of lung lesions expressed in %. Groups: endobronchial (n = 7, green) and intranasal (n = 7, orange). **P < 0.01, ***P < 0.001, Mann–Whitney test. Figures 6, 7 show examples of TB lesions developed in (Figure 6H) while minimal or absent in the EB group (Figure 6D). representative IN- and EB-challenged animals in the head Microscopically, the lesional patterns observed were equivalent and lungs, respectively. The longitudinal section of the head in both groups, differing only in their extension and location. of intranasally challenged goats evidenced bilateral extensive On the other hand, the rendered images of the lungs obtained multifocal to coalescent granulomatous lesions in the nasal by CT disclosed more extensive lung lesions and a higher total cavity of 4/7 goats; the endoturbinates and the middle nasal volume of lung mineralization in the EB-challenged animals concha (on the caudal aspect of the nasal cavity) were always (Figure 7). the most affected, while the dorsal and ventral nasal concha Figure 8 presents the distribution of TB lesions in all the and the nasal septum were more variably involved (Figure 6E). animals of each group. The animals of the EB group have minimal Microscopically, granulomas of variable extension were observed involvement in the head structure whereas all animals of the IN in the nasal turbinates (Figure 6F) and nasopharyngeal mucosa group had lesions in the head (all of them in the retropharyngeal (Figure 6G), while the EB-challenged animals did not present LNs). On the other hand, all animals of the EB group had lesions visible lesions in this localization (Figures 6A, B). Lesions in in the thorax but also in the abdominal cavity (especially in the the head lymph nodes were more severe in the IN group spleen), indicative of early hematogenous generalization. Frontiers in Microbiology 06 frontiersin.org Melgarejo et al. 10.3389/fmicb.2023.1236834 FIGURE 5 Post-mortem results in respiratory lymph nodes (LN). (A) The volume of lesions in pulmonary LN (tracheobronchial and mediastinal) expressed in cm3 . (B) M. caprae DNA load in pulmonary LN, measured by quantitative PCR and expressed as Log10 CFU equivalents. (C) The volume of lesions in retropharyngeal LN expressed in cm3 . Groups: endobronchial (EB, green)-challenged and intranasal (IN, orange)-challenged. *P < 0.05, **P < 0.01, ***P < 0.001, Mann–Whitney test. 4. Discussion The results showed that IN infection slowed the progression of the disease in comparison with the EB route in terms of Since vaccination of cattle against TB is currently forbidden in cell-mediated immune response kinetics (antigen-specific IFN-γ the EU due to its interference with the current diagnostic tools responses) and the onset of clinical signs (body weight gain, rectal used in eradication programs, the goat represents an excellent temperature, and respiratory signs). The delay of proinflammatory translational model to investigate the effect of vaccines against TB, responses (determined by IFN-γ levels) in the IN group may which can be tested under field conditions (Pérez de Val et al., be explained by a slower development of granulomatous lesions 2012, 2013; Bezos et al., 2017; Arrieta-Villegas et al., 2018; Roy that normally show a direct correlation with antigen-specific et al., 2018; Melgarejo et al., 2022). Additionally, goats are also a IFN-γ responses (Vordermeier et al., 2002; Pérez de Val et al., suitable animal model for human TB because they resemble more 2012). The kinetics and intensity of proinflammatory responses closely than small laboratory animals and possess key anatomical in the IN group are consistent with the more progressive, less features such as the complex bronchial tree (Mclaughlin et al., intense, and delayed rectal temperature peaks (weeks 5–7 p.c in 1961) and lung parenchyma organized in a net of interlobular the IN group instead of weeks 2–4 p.c. in the EB group). The septa that tend to encapsulate the lesions (Gil et al., 2010), the delayed seroconversion rates (MPB83) or absence of detectable respiratory tract and lung vascularization (Magno et al., 1990), humoral responses (P22) in most animals of the IN group are and the respiratory lymphoid tissues (Liebler-Tenorio and Pabst, also indicative of a slower progression of the disease. The lower 2006). intensity of MPB83-specific IgG responses in the IN group can The present study aimed to evaluate the IN nebulization of also be associated with a lower severity of TB lesions and bacterial M. caprae as a field-like infection entrance pathway. The rationale burden, as previously described in cattle and goats (Lyashchenko was to better reproduce the features of the natural progression et al., 2004; Pérez de Val et al., 2011). of TB infection but in a controlled manner, in order to improve However, at the end of the study, all IN-challenged animals the monitoring of clinical, immunological, bacteriological, and showed TB-compatible clinical signs indicating that the infection pathological parameters that could be used as predictors of vaccine was well-established. This was also supported by the fact that the efficacy in further studies. The new approach was compared with cumulative body weight gains of the IN and EB groups tended to the well-established experimental infection through endobronchial converge at the end of the experiment. Previous studies, carried inoculation (Pérez de Val et al., 2011). The severity and the out by our research group, demonstrated that body weight increase progression of the disease were not only compared by the different in goat kids, experimentally challenged with M. caprae, inversely distribution of lesions in the EB and IN groups but also they were correlates with pathological TB outcome (Melgarejo et al., 2022). holistically evaluated considering all the parameters mentioned From the pathological point of view, the EB infection induces above, according to previous investigations on TB pathogenicity in faster disease progression with the early development of large- goats (Pérez de Val et al., 2013; Domingo et al., 2014). extensive pulmonary lesions similar to those found in certain Frontiers in Microbiology 07 frontiersin.org Melgarejo et al. 10.3389/fmicb.2023.1236834 FIGURE 6 Pathological features in the head. (A–D) Representative images of goats challenged by the endobronchial route (EB). (E–H) Representative images of goats challenged by the intranasal route (IN). (A, E) Longitudinal section of the head. Granulomatous lesions are marked with arrows in the IN-challenged goat. (B, F) Transversal histological section of a normal nasal turbinate compared with an enlarged edematous nasal turbinate with multifocal granulomatous lesions (arrows). Hematoxylin and Eosin stain (H&E), bars 1,000 µm. (C, G) Nasopharyngeal mucosa with mucosal-associated lymphoid tissue. Noticeable multiple granulomas (arrows) in the IN animal in G. H&E, bars 1,000 µm. (D, H) Tuberculous lesions in the retropharyngeal lymph nodes of the EB-challenged group were absent or minimal as the one shown in the insert in (B), or extensive and obliterating the whole lymph node in the IN group as shown in H. H&E, bars 1,000 µm. Frontiers in Microbiology 08 frontiersin.org Melgarejo et al. 10.3389/fmicb.2023.1236834 FIGURE 7 Tuberculosis lesions evidenced by computed tomography (CT) scan in the lungs. (A–C) Goat challenged by the endobronchial route. (D–F) Goat challenged by the intranasal route. (A, D) CT scan image showing the tracheobronchial tree and TB lesions. (B, E) CT-rendered image of TB lesions (whitish) on the total volume of the lung (dark gray). (C, F) CT-rendered image of mineralization (red) on the total volume of the lung (dark gray). FIGURE 8 Distribution of TB lesions. Blue and red cells indicate the absence and presence of lesions, respectively. (*) In the case of RF LN, pulmonary LN, and lungs, a gradation of red levels represents the quintile distribution of volumes of lesions or number of affected lobes (scale: L -Low: 1- to H -High: 5-). Groups: endobronchial (EB)-challenged (N = 7) and intranasal (IN)-challenged (N = 7). LN, lymph node; SM, submandibular; RF, retropharyngeal; PL, pulmonary; HP, hepatic; MS, mesenteric. Frontiers in Microbiology 09 frontiersin.org Melgarejo et al. 10.3389/fmicb.2023.1236834 chronic advanced cases of caprine TB in the field (Domingo et al., 2018; Melgarejo et al., 2022). This phenomenon could also have 2014). In contrast, after 9 weeks, goats infected through the IN occurred in two IN-challenged animals that showed lesions in the route showed moderate pulmonary lesions similar to those usually spleen (suggestive of hematogenous dissemination), one of which obtained in natural infections, characterized by small to medium, had the most extensive lung TB lesions in the IN-challenged group, multifocal lung nodular granulomatous lesions with a varying without nasal cavity affectation. degree of encapsulation, variable presence of satellite granulomas, To properly develop a suitable infection model to measure and occasionally, bigger coalescing lesions with central cavitation. the efficacy of TB treatments, it would be necessary to refine the These lung lesions are often associated with lesions in the thoracic nebulization procedure to ensure that most of the inoculum enters lymph nodes (Domingo et al., 2014). The main drawback of the into the trachea, avoiding the biased involvement of the nasal IN route is the intense granulomatous lesions found in the nasal cavity. This could be accomplished by introducing the cannula cavity and head LN in some animals, which were artificially induced directly into the ventral nasal meatus. Another explanation for the by the nebulization platform used to inoculate the mycobacteria. skewed distribution of lesions in the IN-challenged goats could be These lesions are not usually observed in field cases (Sanchez et al., the fact that the animals might not have inhaled normally when 2011). This might be explained by the fact that during the challenge, inoculated and even tried to reject or swallow the inoculum. Thus, a considerable proportion of the droplets containing mycobacteria sedation of the goats before the experimental challenge could also bulk delivered into the nasal cavity was not inhaled and remained facilitate a more direct delivery to the lungs. in the upper airways, thus generating local granulomatous lesions. To date, different approaches to MTBC experimental infections In contrast, airborne aerosols transporting mycobacteria in field have been modelized in goats: transthoracic (Bezos et al., 2010, conditions are likely to access the lung parenchyma directly and 2015), endobronchial (Pérez de Val et al., 2011; Wedlich et al., in discrete installments, thus bypassing the upper airways (Daniel 2022), aerosolization (Gonzalez-Juarrero et al., 2013), and the et al., 2009). infection by contact approach, i.e., the exposure of susceptible goats In an experimental infection conducted in calves inoculated to infected ones by close contact (Bezos et al., 2017; Roy et al., intranasally with M. bovis, most animals (11/12) developed TB 2019). The latter has the advantage to mimic natural infection in granulomatous lesions in LNs associated with the upper respiratory the herd but has the disadvantage of not controlling critical points tract, and only four of them had lesions in the lungs or such as the infection time point, infection dose, and infection route, tracheobronchial LN (McCorry et al., 2005). In the present study, which hinder the monitorization of the infection progress and, we investigated not only the head LNs but also performed a most critically, comparison between treatment groups. Similarly, longitudinal section of the skull to investigate the nasal cavity. the use of aerosolizers enables the inhalation of mycobacteria We found macroscopic intranasal granulomas in 4/7 animals that (Gonzalez-Juarrero et al., 2013) but does not allow to deposit the partially obstructed the upper airways and, in one case, provoked inoculum in a defined site of the respiratory tract at the start- severe dyspnea that led to the humane euthanasia of one animal at point of the experiment, to compare the pathological evolution week 8 before the completion of the experiment. Putative variations from the infection site among the treatment groups. The atomizing of the location of the cannula in the dorsal or middle nasal meatus device used in the present study sprays droplets > 30 mm in vs. the ventral nasal meatus could explain the location of lesions diameter that resemble those excreted by sneezing or coughing. in the caudal aspect of the nasal cavity in 4/7 animals, while in With the IN nebulization one can control the infection time point the 3/7 animals with no lesions, the cannula was likely introduced and infection dose conditions but still not ensure the infection in the ventral meatus. In one of these animals, the lesions in the site within the respiratory tract, resulting in moderate pulmonary lung were the most severe in the group (animal 179, suggestive TB lesions in only 4/7 animals, probably because a significant of an effective delivery of the inoculum to the lung), while the fraction of the inoculum was retained in the nasal cavity, whereas other two only developed lesions in the retropharyngeal lymph all goats infected by EB inoculation showed pulmonary TB lesions nodes. In addition, two animals of the IN group had lesions in as previously described in all experimental infections of goats with the mesenteric LN, and this could be explained by the partial M. caprae or M. bovis by this route (Pérez de Val et al., 2011, ingestion of the inoculum (in the case of animal 153, no lesion 2012, 2013; Arrieta-Villegas et al., 2018, 2020; Melgarejo et al., was observed in the nose), by swallowing the mycobacteria from 2022; Wedlich et al., 2022). However, the EB model, as does the granulomatous lesions developing in the nasal cavity and/or the transthoracic one, can induce advanced caseous-necrotizing associated lymph nodes (in the case of animal 226) or, less likely, lesions even in vaccinated individuals, limiting the capacity to due to lymphohematogenous dissemination to the abdominal predict the protective effect of vaccinated candidates (Arrieta- cavity. The involvement of mesenteric LN was a frequent finding in Villegas et al., 2020). In contrast, the IN infection induced low-to- natural settings in a field study carried out in a goat herd, where the moderate pulmonary lesions with a low degree of mineralization, most likely transmission pathway was the shared feeders between suggesting a more initial developmental stage that may facilitate infected and susceptible animals (Vidal et al., 2017). Spread of TB the evaluation of the containment of TB lesions spreading in lesions beyond the upper airways and thoracic cavity also occurred immunized animals. Optimization of the model is required to reach in all EB-challenged animals (i.e., all of them showed lesions in the a compromise among (A) conditions control (dose, inoculation spleen), most likely due to a lymphohematogenous spreading from point), (B) duration of the experiment, and (C) reproducibility of the primary complex generated in the lungs and pulmonary LN, TB field phenotype in order to precisely evaluate the efficacy of the similar to that reported in previous studies (Arrieta-Villegas et al., tested vaccines. Frontiers in Microbiology 10 frontiersin.org Melgarejo et al. 10.3389/fmicb.2023.1236834 Data availability statement Funding The original contributions presented in the This study was funded through a grant from the Spanish study are included in the article/supplementary Ministry of Science and Innovation (Ref. PID2019-105155RB- material, further inquiries can be directed to the C32/AEI/10.13039/501100011033). IRTA was supported by corresponding authors. CERCA Programme/Generalitat de Catalunya. CM is the recipient of a pre-doctoral grant from the program Don Carlos Antonio López of the Republic of Paraguay (Ref. 88/2020). Ethics statement Acknowledgments The animal studies were approved by Animal Welfare Committee of the Generalitat de Catalunya. The studies The authors are grateful to Abel Muñoz, Mónica Pérez, Rosa were conducted in accordance with the local legislation and López, Anna Barceló, and the staff of the BLS-3 Unit of IRTA- institutional requirements. Written informed consent was CReSA for their outstanding technical support. not obtained from the owners for the participation of their animals in this study because the animals were purchased for these studies. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be Author contributions construed as a potential conflict of interest. BP and EV conceived and planned the study. CM, AC, CP, MM, ZC, GC, XM, YE, MD, EV, and BP performed the Publisher’s note experiments and contributed to data collection. CM, AC, JF, CP, YE, EV, and BP analyzed and interpreted the data. BP and MD All claims expressed in this article are solely those of the acquired funding and were in charge of project administration. authors and do not necessarily represent those of their affiliated CM, EV, and BP wrote the original draft. 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