Influence of heterologous and homologous vaccines, and their components, on the host immune response and protection against experimental caprine paratuberculosis

ORIGINAL RESEARCH published: 05 January 2022 doi: 10.3389/fvets.2021.744568 Frontiers in Veterinary Science | www.frontiersin.org 1January 2022 | Volume 8 | Article 744568 Edited by: Francisco Javier Salguero, Public Health England, United Kingdom Reviewed by: Beatriz Vidana, University of Bristol, United Kingdom Mourad Tayebi, Western Sydney University, Australia *Correspondence: Noive Arteche-Villasol [email protected] †These authors have contributed equally to this work Specialty section: This article was submitted to Veterinary Experimental and Diagnostic Pathology, a section of the journal Frontiers in Veterinary Science Received: 20 July 2021 Accepted: 02 December 2021 Published: 05 January 2022 Citation: Arteche-Villasol N, Gutiérrez-Expósito D, Elguezabal N, Sevilla IA, Vallejo R, Espinosa J, Ferreras MC, Benavides J and Pérez V (2022) Influence of Heterologous and Homologous Vaccines, and Their Components, on the Host Immune Response and Protection Against Experimental Caprine Paratuberculosis. Front. Vet. Sci. 8:744568. doi: 10.3389/fvets.2021.744568 Influence of Heterologous and Homologous Vaccines, and Their Components, on the Host Immune Response and Protection Against Experimental Caprine Paratuberculosis Noive Arteche-Villasol1,2*†, Daniel Gutiérrez-Expósito1,2†, Natalia Elguezabal3, Iker A. Sevilla3, Raquel Vallejo1,2, José Espinosa1,2, María del Carmen Ferreras1,2, Julio Benavides2and Valentín Pérez1,2 1Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad de León, León, Spain, 2Departamento de Sanidad Animal, Instituto de Ganadería de Montaña (CSIC-ULE), León, Spain, 3Departamento de Sanidad Animal, NEIKER-Instituto Vasco de Investigación y Desarrollo Agrario, Derio, Spain Vaccination against paratuberculosis, a chronic disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map), has been considered as the most effective control method. However, protection is incomplete, and the mechanisms operating in the response of the animals to vaccination are not fully understood. Therefore, this study analyzed the immune response and the effects on protection against Map infection, elicited by paratuberculosis (Silirum®) and tuberculosis (heat-inactivated M. bovis [HIMB]) vaccines and their components in a caprine experimental model. Fifty goat kids were divided into 10 groups (n=5) according to their vaccination (Silirum®, HIMB and nonvaccinated), immunization (inactivated bacteria or adjuvant), and/or infection. Oral challenge with Map was performed 45 days postvaccination/immunization (dpv), and animals were euthanized at 190 dpv. Peripheral immune response and proportion of lymphocyte subpopulations were assessed monthly by enzyme-linked immunosorbent assay and flow cytometry analysis, respectively. Local immune response, proportion of tissue lymphocyte subpopulations, Map detection (polymerase chain reaction), and histological examination were conducted in gut-associated lymphoid tissues. All infected groups developed paratuberculosis granulomatous lesions despite vaccination or immunization. The Silirum®and HIMB-vaccinated groups showed a considerable lesion reduction consistent with a significant peripheral cellular and humoral immune response. Besides, a lower number of granulomas were observed in groups immunized with inactivated bacteria and adjuvants in comparison to nonvaccinated and infected group. However, despite not being significant, this reduction was even higher in adjuvant immunized groups, which developed milder granulomatous lesion with no detectable peripheral immune responses associated with immunization. No changes in the peripheral and local proportion of lymphocyte subsets or local immune response were detected in relation to either vaccination/immunization or infection. Arteche-Villasol et al. Vaccination Against Caprine Paratuberculosis Despite that paratuberculosis and tuberculosis vaccination showed a partial and cross-protection against Map infection, respectively, only histological examination could assess the progression of infection in these animals. In addition, the pattern observed in the reduction of the lesions in adjuvant immunized groups suggests the possible involvement of a nonspecific immune response that reduces the development of granulomatous lesions. Keywords: vaccination, paratuberculosis, tuberculosis, HIMB, adjuvant, cross-protection INTRODUCTION Paratuberculosis is a chronic and debilitating disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map) (1) and responsible for significant global economic losses estimated at approximately US $12.61 million and $364.31 million in Spanish and European Union dairy cattle, respectively (2,3). Map is a ubiquitous bacterium transmitted among the livestock at an early age through the fecal–oral route, although clinical manifestations may appear several years after infection (4). Initial protective immune response against Map has been related to a strong cell-mediated immune response characterized by the release of the proinflammatory cytokine interferon γ(IFN-γ), the formation of granulomas, and the clearance of the mycobacteria (5,6). In this sense, Map-infected animals can show a variety of lesions that have been shown to be closely related to the different phases of the disease. Early paratuberculosis lesions are characterized by small and well-demarcated granulomas, named focal or multifocal forms, located within the intestinal lymphoid tissue that can be also seen in adult animals, where they are considered as forms of latency or resistance; disruption of the protective cell-mediated immune response leads to the development of diffuse lesions and the evolution toward a widespread granulomatous enteritis and the occurrence of clinical signs (7,8). Pathological methods for paratuberculosis lesion characterization have been successfully applied in previous works as a reliable indicator of the presence of Map infection and the form of the disease shown by the infected animals (9–11). The long incubation period and the ability of Map to persist in the environment hamper control programs based on hygienemanagement measures or early diagnosis and culling of positive animals (12,13). For this reason, vaccination with commercially available heat-killed vaccines has been considered as the most cost-effective measure for the control of paratuberculosis, as its use reduces the incidence of the disease within the herd (14,15). Vaccination leads to a reduction of both the colonization of intestinal tissues and the number of clinically affected animals, achieving a decrease by approximately 90% in the occurrence of Map-shedding animals (16–18). However, the mechanisms that might be involved in the protection associated with vaccination are yet not fully understood (12,13). Protection conferred by vaccines against Map infection has been correlated with an early and strong cell-mediated immune response (19–21), despite the fact that there is a low percentage of vaccination failure, where some vaccinated animals could remain highly infectious and develop severe lesions (16,22,23). Most paratuberculosis heat-killed vaccines are based on Map 316F strain, which has demonstrated reduced virulence (tissue bacterial burden) during experimental infection of calves with this live isolate likely due to continuous in vitro passages (24). In this sense, subcutaneous immunization of sheep with heatkilled Map 316F strain alone did not show significant cellmediated and humoral immune response (25). Supplementation with mineral oil adjuvants is a core principle in order to enhance the immunogenicity of antigens and their continuous liberation in order to promote a robust immune response through the mount of a strong local inflammation at the site of injection (26). In a previous experiment, significant variations in the immune response were found when paratuberculosis vaccines made with different adjuvants were subcutaneously administered to sheep (25). On the other hand, the sole administration of adjuvants has been shown to elicit an unspecific immune response whose protective effect has not been evaluated in detail (27–29). In the case of paratuberculosis vaccines, the effect that each component could have in a protective response against Map infection has not been fully elucidated. Besides, Map shares a high number of common antigens with related mycobacteria such as Mycobacterium bovis (Mbv) or Mycobacterium caprae, responsible for tuberculosis in ruminants, a fact that is beyond the cross-reactions that appear when immunological-based tests are used for tuberculosis diagnosis in paratuberculosis infected or vaccinated animals (30,31). This also could be associated with the significant reduction of tuberculosis-related lesions achieved after paratuberculosis vaccination in goats and calves (32,33). Despite this fact, the effect of immunization with tuberculosis-related mycobacteria on the development of paratuberculosis is yet unknown. Therefore, the objective of this study was to analyze the effect of homologous or heterologous vaccination of goats, and a subsequent challenge with Map, on the immune response and protection and evaluate whether the different components from these vaccines (inactivated bacteria or adjuvant) could participate in the response of the vaccinated host against Map. MATERIALS AND METHODS Ethics Statement All the experimental procedures were carried out according to European (86/609) and Spanish laws (R.D. 223/1988, R.D. 1021/2005, R.D. 53/2013) and approved by the local government Frontiers in Veterinary Science | www.frontiersin.org 2January 2022 | Volume 8 | Article 744568 Arteche-Villasol et al. Vaccination Against Caprine Paratuberculosis following the report and suggestions from the Subcommittee on Animal Experiments and Welfare of the University of León (ULE) (OEBA-ULE-016-2017). Vaccines, Immunization Products, and Challenge Inoculum Silirum R commercial vaccine against paratuberculosis and its components (the MontanideTM adjuvant and the Map 316F strain included in the vaccine) were prepared separately and shipped by the manufacturer, CZ Vaccines (Porriño, Spain), whereas the heat-inactivated Mbv vaccine (HIMB) and its components (a MontanideTM adjuvant and the 1403 Mbv strain) were prepared by NEIKER as previously described (34). Briefly, each dose of HIMB vaccine (1 mL) consisted of an aqueous suspension of heat-inactivated 107colonyforming units (CFUs) of Mbv 1403 NEIKER’s strain (84–85◦C for 45 min) emulsified in MontanideTM ISA 50 V 2 adjuvant (Seppic, France). Furthermore, each inactivated bacteria and adjuvant immunization products were adjusted to the same dose administered in Silirum R and HIMB vaccines. Thus, each dose (1 mL) of inactivated bacteria (109CFUs of Map 316F and 107 CFUs of Mbv 1403 strains) was diluted in phosphate-buffered saline (PBS), whereas each dose of MontanideTM adjuvant (1 mL) (used in Silirum R and HIMB vaccines) was emulsified in PBS. Besides, bovine Map 764 strain was prepared for the challenge as previously described by Fernández et al. (11). Briefly, Map 764 strain was grown on Middlebrook 7H9 broth enriched with 10% oleic acid–albumin–dextrose–catalase (OADC) and 2 mg · L−1Mycobactin J (7H9 OADC MJ) for 3 weeks at 37 ±1◦C. Then, cultures were harvested by centrifugation at 3,000gfor 10 min, and bacterial pellets were washed twice and resuspended in PBS. In order to disrupt bacterial clumps, resultant suspension was passed up and down through a 27-gauge needle several times and vortexed. Bacterial concentration was estimated by optical density (O.D.) and CFU estimation of 10-fold serial dilutions plated onto agar-solidified 7H9 OADC MJ. Finally, suspensions were adjusted to 1.2 ×1010 CFUs ·mL−1and maintained at 4◦C throughout all the challenge period (2 weeks), and bacterial clumps were again disrupted before oral inoculation as mentioned previously. Experimental Design A total of 50 female Murciano–Granadina breed goat kids of age 1 month were used in this study. Animals were selected from a flock without clinical signs and tested negative to paratuberculosis and tuberculosis in the last 10 years. Moreover, no positive reactors were identified in the annual official tuberculosis eradication campaigns, based on intradermal skin test, conducted by the regional animal health authorities during the last 5 years. The paratuberculosisand tuberculosis-free status of the experimental animals was confirmed using antibody enzyme-linked immunosorbent assay (ELISA) against Map (ID Screen R Paratuberculosis indirect, IDVet, Gabrels, France) and Mbv (INgezim Tuberculosis DR, Eurofins Technology, Madrid, Spain) and the IFN-γrelease test (Bovigam R Mbv IFN-γtest for cattle, Thermo Fisher Scientific, Waltham, USA). After an adaptation period of 15 days in the facilities of the Instituto de Ganadería de Montaña (IGM-ULE) in León (Spain), goat kids were randomly allocated in separated pens, subjected to standard management practices, and their health and welfare status was checked daily. At the beginning of the study, and according to the vaccination and/or infection protocol to be followed, goats were classified into 10 groups (n=5) and distributed in different pens in order to prevent direct contact between groups according to the following scheme (Figure 1): VS: Silirum R vaccinated and noninfected VSI: Silirum R vaccinated and infected VH: HIMB vaccinated and noninfected VHI: HIMB vaccinated and infected StrSI: immunized with Map 316F Silirum R strain and infected StrHI: immunized with Mbv 1403 HIMB strain and infected AdjSI: immunized with MontanideTM Silirum R adjuvant and infected AdjHI: immunized with MontanideTM HIMB vaccine adjuvant and infected NV: nonvaccinated and noninfected NVI: nonvaccinated and infected No uninfected animals were included in groups immunized with inactivated bacteria or adjuvants, because of the fact that the study was focused on the evaluation of the effect of the components of the vaccines on protection, after Map infection, and this would involve the inclusion of a large number of animals and groups that would hinder the study. Vaccination was performed at the beginning of the experiment, on day 0, by a subcutaneous injection in the brisket with 1 mL of Silirum R vaccine, 109Map 316F Silirum R  vaccine strain CFUs, 1 mL of MontanideTM Silirum R adjuvant (CZ Vaccines, Porriño, Spain), 1 mL of HIMB vaccine, 107 Mbv 1403 HIMB vaccine strain CFUs, and MontanideTM HIMB vaccine adjuvant, whereas 10 animals remained as the control group (nonvaccinated) and were inoculated subcutaneously with 1 mL of PBS (Figure 1). Forty-five days postvaccination (dpv), animals were challenged orally (Figure 1) using an automatic syringe with a total amount of 1.2 ×1010 Map 764-CFUs diluted in 40 mL of PBS as previously described by Fernández et al. (11), whereas 40 mL of PBS was administered orally to noninfected animals at the same time. Throughout the experimental trial, all goats were monitored daily for clinical signs and sampled every 30 days until sacrifice (Figure 1). At 190 dpv, complete necropsies and postmortem sampling were performed on all goats after being humanely euthanized by deep sedation with xylazine (XILAGESIC R ,Laboratorios Calier, Barcelona, Spain) and a subsequent intravenous injection of T61 R (MSD Animal Health, Salamanca, Spain) followed by exsanguination (Figure 1). Sample Collection Blood samples were monthly collected from the jugular vein into Vacutainer tubes with lithium heparin (Becton Dickinson and Company, UK) and without anticoagulant (Becton Dickinson and Company, UK). Then, heparinized samples were processed immediately for IFN-γrelease test in response to protein derivative of avian (PPDa) and bovine (PPDb) antigens at 0, Frontiers in Veterinary Science | www.frontiersin.org 3January 2022 | Volume 8 | Article 744568 Arteche-Villasol et al. Vaccination Against Caprine Paratuberculosis FIGURE 1 | Experimental design scheme. Goats were divided into seven groups and vaccinated with different components at 0 days postvaccination (dpv). From then until sacrifice (190 dpv), blood samples (B) were taken at 30-day interval. Five goats from each group were orally challenged with Map 764 strain at 45 dpv and divided into 10 groups: NV, nonvaccinated and noninfected; NVI, nonvaccinated and infected; VS, Silirum®vaccinated and noninfected; VSI, Silirum®vaccinated and infected; VH, HIMB vaccinated and noninfected; VHI, HIMB vaccinated and infected; StrSI, Map 316F Silirum®strain immunized and infected; StrHI, Mbv 1403 HIMB strain immunized and infected; AdjSI, MontanideTM Silirum®adjuvant immunized and infected; AdjHI, MontanideTM HIMB adjuvant immunized and infected. Finally, at 190 dpv, feces (F) were collected, and culling and complete necropsies for tissue (T) sample collection were carried out. 30, 60, 90, 120, 150, and 190 dpv (Figure 1). At the same time points, heparinized blood was also collected for peripheral blood mononuclear cell (PBMC) isolation (35) and their subsequent characterization by flow cytometry. Besides, nonheparinized samples were processed for Map (ID Screen R Paratuberculosis indirect, IDVet, Gabrels, France) and Mbv-specific (INgezim Tuberculosis DR, Eurofins Technology, Madrid, Spain) antibody determination tests at 0, 30, 60, 90, 120, 150, and 190 dpv (Figure 1). Fecal samples from each goat were collected separately into disposable plastic gloves directly from the rectum at 190 dpv, prior to the euthanasia of the animal, and frozen at −20◦C until processing for Map detection through bacteriological culture. Animals were euthanized at day 190 dpv and a regulated, orderly, and complete necropsy was performed. After gross examination of the viscera, samples from ileum (proximal, medium, and distal zones), jejunum (proximal, medium, and distal zones), ileocecal valve, and jejunal Peyer patches (at least three patches from each zone: proximal, medium, and distal), together with mesenteric, jejunal, and ileocecal lymph nodes, were taken into buffered formol saline fixative for histological examination. In addition, samples of distal ileum, jejunal Peyer patches, and mesenteric lymph node were also collected and stored at −20◦C for Map isolation by culture and detection by real-time quantitative polymerase chain reaction (qPCR) as well as processed immediately for leukocyte isolation in order to characterize lymphocyte subpopulation by flow cytometry and also to perform IFN-γrelease test in response to PPDa and PPDb antigens. Isolation of PBMCs and Tissue Leukocytes PBMCs were isolated as previously described (35). Briefly, 30 mL of heparinized peripheral blood was centrifuged, and PBMCs were isolated by gradient centrifugation using LymphoprepTM (STEMCELL Technologies R , Cologne, Germany). Resultant PBMCs were washed three times with PBS, and cell suspensions were resuspended in supplemented RPMI1640 medium + GlutaMaxTM (Gibco, Paisley, UK) and counted in a Neubauer chamber and adjusted at a final concentration of 106cells · mL−1. Intestinal samples and mesenteric lymph nodes were collected and washed in PBS during the necropsy, put into individual falcon tubes containing 30 mL of sterile supplemented RPMI1640 medium, and processed in the laboratory within 30 min after collection. For tissue lymphocyte isolation, 5 cm of ileum and jejunal Peyer patches were longitudinally opened showing the mucosa and washed with PBS until fecal remains were eliminated, whereas pericapsular fat was removed from the lymph nodes. The excess tissue around each Peyer patch was removed, and the mucosa from ileum and Peyer patches were scraped and minced. Besides, 50 mg of mesenteric lymph node tissue was cut into small pieces and chopped using a scalpel blade. Minced tissue was suspended in 11 mL of PBS with EDTA (2 mM) and processed with a stomacher blender (Masticator, IUL) for 2.5 min. Then, 10 mL from the upper homogenized portion was passed through 40-µm filter (Thermo Fisher Scientific, Madrid, Spain), and resultant suspension was layered in an equal volume of LymphoprepTM and centrifuged at 800 gfor 30 min with no stop or acceleration. Cells from the interface layer were washed three times with PBS/EDTA, counted in a Neubauer chamber and Frontiers in Veterinary Science | www.frontiersin.org 4January 2022 | Volume 8 | Article 744568 Arteche-Villasol et al. Vaccination Against Caprine Paratuberculosis resuspended in supplemented RPMI1640 at a final concentration of 106cells ·mL−1. Cell viability determined by trypan blue dye exclusion was usually >90% for both PBMCs and tissue lymphocytes (data not shown). Peripheral and Local Cell-Mediated and Peripheral Humoral Immune Response Whole heparinized peripheral blood samples taken at 0, 30, 60, 90, 120, 150, and 190 dpv were processed within 3 h of collection. For each animal, three wells of a 24-well tissue plate (Thermo Fischer Scientific, Rochester, NY) were filled with 1.5 mL of blood each and were either mixed with 100 µL of sterile PBS (negative control) or stimulated with 100 µL of PPDa or PPDb (CZ Vaccines, Porriño, Spain) at a final concentration of 30 µg· mL−1diluted in sterile PBS (11). After 22 h of stimulation, plates were centrifuged at 750 gfor 15 min, and plasma was collected and stored at −20◦C until tested. For the analysis of the cell-mediated local immune response, a total of 2 ×106mononuclear leukocytes isolated from ileum, Peyer patches, and mesenteric lymph node were seeded per well into three wells each of a 24-well tissue plate and stimulated as described for whole blood. After 22 h of stimulation, plates were centrifuged at 750 gfor 15 min, and supernatants were collected and stored at −20◦C until tested. IFN-γproduction in these assays was assessed by duplicate using commercial ELISA for bovine IFN-γfollowing manufacturer’s instructions (Bovigam R Mbv IFN-γtest for cattle, Thermo Fisher Scientific, Waltham, USA), and absorbance values were measured spectrophotometrically using an ELX800 ELISA reader (Bio-Tek Instruments) at 450 nm. The O.D. values were adjusted by dividing the plasma or supernatant O.D. value by the negative control O.D. value of each plate in order to prevent interplate variations. Results were expressed as avian and bovine index values by means of the quotient between the mean O.D. of PPDa or O.D. of PPDb-stimulated plasma, respectively, and the mean O.D. of the negative control plasma (11,36). Nonheparinized blood samples were allowed to clot and retract, and serum was stored at −20◦C until used. Each serum was tested for specific antibodies against Map (37) and Mbv (38) using commercial ELISA tests (ID Screen R Paratuberculosis indirect, IDVet, Gabrels, France; and INgezim Tuberculosis DR Eurofins Technology, Madrid, Spain, respectively) following manufacturer’s instructions. Results were expressed as the S/P ratio of Map and Mbv antibodies calculated by dividing the corrected O.D. of the sample by the corrected O.D. of the positive control and multiplying by 100 (39). Real-Time Quantitative Detection of Map DNA was extracted from 50 mg of distal ileum, jejunal Peyer patches, and mesenteric lymph node by using Maxwell R 16 Tissue DNA Purification Kit (Promega, WI, USA) with the Maxwell 16 Instrument (Promega) following manufacturer’s instructions. Thereupon, DNA was quantified using QuantiFluorTM ONEdsDNA System kit (Promega, WI, USA) and QuantusTM Fluoremeter (Promega, WI, USA). Extracted DNA was diluted at 50 ng ·µL−1and stored at −20◦C until qPCR was performed. Genomic DNA from 2×108Map CFUs and that from 50mg of tissues of a noninfected animal were extracted and quantified to generate a standard curve. Detection of Map IS900 sequence was performed as previously described by Arteche-Villasol et al. (40). In addition, MapDNA quantification and qPCR analytical sensitivity were assessed by the construction of a 10-fold diluted standard curve using Map-genomic DNA ranging from 1,000 pg to 0.001 pg/reaction mixed with 100 ng/reaction of tissue DNA from a noninfected animal. Samples were considered as positive when the dissociation peak (Tm) was 89.1 ±1.5◦C and threshold cycles (Ct) were ≤37 (41,42). The qPCR results were analyzed using 7500 Software v2.0.6 (Applied BiosystemsTM). Map-DNA quantity (pg) of each well was calculated by interpolation of their Ct values with the standard curve as previously described (43), and the mean quantity was calculated from both duplicates. Tissue and Fecal Culture of Map Distal ileum, jejunal Peyer patches, and mesenteric lymph node from each animal were tested individually, whereas fecal samples were pooled into groups of three goats each. Ileum and Peyer patch segments (12 cm) and 2 grams of mesenteric lymph node were processed as described for tissue leukocyte isolation and following methods previously described (44). Briefly, 2 g of each tissue and fecal samples pools were decontaminated with 38 mL of hexadecylpyridinium chloride and homogenized in a stomacher blender (Masticator, IUL) for approximately 15 s. After 18 h of decontamination, 200 µL of the suspension was used to inoculate two tubes containing Herrold’s egg yolk medium supplemented with sodium pyruvate and mycobactin J (MJ) and two tubes containing 7H9 OADC supplemented with MJ, penicillin, amphotericin, and chloramphenicol. Cultures were incubated at 37◦C±1◦C, and growth was checked by examination under a stereoscopic microscope after 8, 12, 16, and 20 weeks postinoculation. Cultures were considered positive if one or more characteristic Map colonies were observed in any tube. Colonies isolated on both mediums were confirmed by a real-time multiplex PCR detecting IS900 and ISMap02 Map sequences (45). Flow Cytometric Analysis of PBMCs and Tissue Leukocytes Single-color flow cytometry analysis was carried out for phenotypic characterization of PBMCs isolated at 0, 30, 60, 90, 120, 150, and 190 dpv and mononuclear leukocytes isolated from distal ileum, jejunal Peyer patches, and mesenteric lymph node. A total number of 2 ×105cells per well were seeded in a 96well plate (Thermo Fisher Scientific, Roskilde, Denmark) and incubated with primary antibodies against lymphocyte surface markers detailed in Table 1 for 1 h at 4◦C. Afterward, cells were washed twice with PBS and incubated with appropriate conjugated secondary antibodies for 1 h at 4◦C (Table 1). Finally, cells were fixed with 1% of CellFIXTM (Becton Dickinson and Company, Erembodegem, Belgium) until analyzed. Sample Frontiers in Veterinary Science | www.frontiersin.org 5January 2022 | Volume 8 | Article 744568 Arteche-Villasol et al. Vaccination Against Caprine Paratuberculosis TABLE 1 | Primary and secondary antibodies used in flow cytometry analysis of PBMCs and tissue lymphocyte subpopulations. Target Specificity Monoclonal/polyclonal Primary antibody dilution Reference Secondary antibody Secondary antibody dilution CD4 T helper lymphocytes Monoclonal 1:400 MCA2213GA, BioRad Polyclonal rabbit anti–mouse IgG-FITC, F0313, Dako 1:50 CD8 Cytotoxic T lymphocytes Monoclonal 1:400 MCA2216GA, BioRad Polyclonal rabbit anti–mouse IgG-FITC, F0313, Dako 1:50 WC1 γδ T lymphocytes Monoclonal 1:200 MCA8586, BioRad Polyclonal rabbit anti–mouse IgG-FITC, F0313, Dako 1:50 CD21 Naive B lymphocytes Monoclonal 1:10 MCA1185, BioRad Polyclonal rabbit anti–mouse IgG-FITC, F0313, Dako 1:50 CD20 Mature B lymphocytes Polyclonal 1:100 9013-P, Thermo Scientific Polyclonal goat anti–rabbit IgG-Alexa Fluor®488, ab150077, Abcam 1:2,000 CD3 T lymphocytes, natural killer Polyclonal 1:100 A0452, Dako Polyclonal goat Antirabbit IgG-Alexa Fluor®488, ab150077, Abcam 1:2,000 acquisition of 10,000 events was performed using a flow cytometer (MACSQuant, Miltenyi Biotec R ), where events were gated during the acquisition analysis as previously described elsewhere (35) to discard the presence of air and doublets. Then, analysis of data was carried out using the MACSQuantify10 SoftwareTM (Miltenyi Biotec R ), and results were expressed as percentage of positive cells. Histopathological Examination Fixed tissues for histopathological examination were conventionally processed for paraffin embedding and stained with hematoxylin–eosin and Ziehl–Neelsen technique for acid-fast bacilli detection (46). Lesions consistent with Map infection were classified as focal,multifocal a and multifocal b, or diffuse forms according to the presence and location of granulomas and following the guidelines previously described for small ruminants (8, 46). Briefly, lesions were characterized as focal forms when granulomas were restricted to the lymphoid tissue of the Peyer patches; multifocal forms when the granulomas were located in the lamina propria adjacent to the lymphoid tissue (multifocal a) or not (multifocal b) and diffuse forms when granulomas spread to wide areas of the mucosa. Classification of each animal was based on its most severe granulomatous lesion. Following histopathological examination, the number of granulomas per tissue section was quantified in all tissue samples as described elsewhere (11,46). Three tissue sections of each intestinal site (i.e., ileum—proximal, medium, and distal zones; jejunum—proximal, medium, and distal zones; ileocecal valve and jejunal Peyer patches—proximal, medium, and distal zones) and two sections from each lymph node (i.e., mesenteric, jejunal and ileocecal lymph nodes), were selected, and the mean number of granulomas per animal was recorded by the same observer (V.P.; diplomat of the European College of Veterinary Pathologists), distinguishing those granulomas located in the lymphoid tissue from those located in the associated lamina propria or in the mucosa not related to lymphoid tissue. Statistical Analysis Normal distribution of the results from Mapand Mbvspecific antibodies, local and peripheral IFN-γproduction, and proportion of peripheral and tissue lymphocyte subpopulations were assessed for normality using Shapiro–Wilk test. Data from IFN-γproduction and antibody ELISA tests were logarithmically transformed. Then, flow cytometry and ELISA results from peripheral IFN-γand antibody production were analyzed using generalized lineal model (GLM) procedure for evaluation of the main effects of vaccination, challenge, time, and its interactions. Subsequently, differences between vaccination groups for each time sampled were estimated using Tukey– Kramer correction for multiple comparisons. Similarly, the main effects of vaccination, tissue, and its interactions were estimated in the results of local IFN-γproduction followed by the evaluation of differences between vaccination groups and tissues using Tukey–Kramer multiple-comparisons test. Besides, differences between groups in the number of goats with lesions were evaluated using χ2and Fisher exact tests. In addition, after logarithmic transformation, differences in the granuloma counts between vaccination groups were calculated using the Student t-test. All statistical analyses were carried out using GraphPad Prism 6.0 software (San Diego, CA, USA) excluding GLMs that were performed using R software 3.5.3 (R Development Core Team, 2019). P<0.05 was considered statistically significant. RESULTS Peripheral and Local Cell-Mediated Immune Response (IFN-γ) Results from GLM showed that avian and bovine index values reached significant levels from 60 to 190 dpv in the VS, VSI, Frontiers in Veterinary Science | www.frontiersin.org 6January 2022 | Volume 8 | Article 744568 Arteche-Villasol et al. Vaccination Against Caprine Paratuberculosis FIGURE 2 | Kinetics of the IFN-γproduction by whole blood stimulated with avian (PPDa) and bovine (PPDb) antigens. Results are expressed as avian (A) and bovine (B) O.D. index values of each vaccination group (n=5) (NV, nonvaccinated and noninfected; NVI, nonvaccinated and infected; VS, Silirum®vaccinated and noninfected; VSI, Silirum®vaccinated and infected; VH, HIMB vaccinated and noninfected; VHI, HIMB vaccinated and infected; StrSI, Map 316F Silirum®strain immunized and infected; StrHI, Mbv 1403 HIMB strain immunized and infected; AdjSI, MontanideTM Silirum®adjuvant immunized and infected; AdjHI, MontanideTM HIMB adjuvant immunized and infected) and time sampled (dpv, days postvaccination). Vertical dotted red line represents the time of Map oral challenge (45 dpv). Significant differences determined by multiple comparisons were represented as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. VH, and VHI groups (p<0.001 and p<0.01 respectively) (Figures 2A,B). In addition, Silirum R (VS and VSI) and HIMB vaccine (VH and VHI) immunization exerted a considerable effect on avian index values regardless of the infection status (p<0.01 and p<0.001, respectively) (Figure 2A), but only the VHI, VH, and StrHI groups showed a significant effect on bovine index values (p<0.01) (Figure 2B). Furthermore, oral challenge significantly impacted on the avian index levels of the VSI, VHI (p<0.001), and NVI (p<0.05) groups and on the bovine index values of the VSI, VHI (p<0.01), and StrHI (p< 0.05) groups. Multiple-comparisons analysis showed that the VSI group had higher avian index values than NV, NVI, StrSI, AdjSI, and AdjHI at 60 and 120 dpv, whereas the VS group showed these differences only at 120 dpv (Figure 2A). In contrast, these groups did not show any significant differences in bovine index levels (Figure 2B). Furthermore, no differences were observed between VS and VSI either in avian or bovine index values at any time point. On the other hand, the VHI group showed greater avian index values than NVI, NV, StrSI, AdjSI, and AdjHI at 120 dpv (Figure 2A). In addition, bovine index values of this group were significantly higher than NV, NVI, StrSI, AdjSI, and AdjHI at 90, 120, 150, and 190 dpv and greater than VH at 120 dpv (Figure 2B). Local IFN-γproduction in mononuclear leukocytes purified from tissues and stimulated with PPDa and PPDb was lower than that observed in PBMCs. Furthermore, avian index values were more heterogeneous than bovine index levels on account of the higher individual variability observed in the former (Figures 3A,B), specifically at the NVI, VH, VHI, StrHI, and AdjSI groups (Figure 3A). Peyer patches and mesenteric lymph node from NVI group showed the highest avian index values followed by the ileum, Peyer patches, and mesenteric lymph node from the StrHI group (Figure 3A). However, statistical analysis showed no significant effect of vaccination or tissue location on the avian or bovine index values (p>0.05). In addition, no significant differences were observed between the vaccinated groups in the multiple-comparisons analysis either (p<0.05). Meanwhile, significant differences were observed in the avian index values within the NVI group, where IFN-γproduction was higher in Peyer patches and mesenteric lymph node than in the ileum (p<0.05) (Figure 3A). Besides, despite the high variability within groups observed in the bovine index values, the values of mesenteric lymph node from the VH group were considerably higher than those of the ileum and Peyer patches (p<0.01) (Figure 3B). Peripheral Humoral Response Results from GLM estimated that both Mapand Mbvspecific antibody production reached significant levels from 60 to 190 dpv (p<0.001 and p<0.0001, respectively) in Silirum R and HIMB-vaccinated groups (Figures 4A,B). In addition, oral challenge boosted Map-specific antibody production in the vaccinated groups VSI, VHI (p<0.001), and NVI (p<0.05) (Figure 4A) and Mbv-specific antibody production in VHI (p<0.001) (Figure 4B). Besides, multiplecomparisons analysis showed that only VSI had significantly higher Map-specific S/P values in comparison with the groups immunized with adjuvants, inactivated bacteria, and nonvaccinated at 90, 120, 150, and 190 dpv, whereas no Frontiers in Veterinary Science | www.frontiersin.org 7January 2022 | Volume 8 | Article 744568 Arteche-Villasol et al. Vaccination Against Caprine Paratuberculosis FIGURE 3 | IFN-γproduction by leukocytes from ileum (IL), jejunal Peyer patches (JPP), and mesenteric lymph node (MLN) stimulated with avian (PPDa) and bovine (PPDb) antigens. Results are expressed as avian (A) and bovine (B) O.D. index values of each vaccination group (n=5) (NV, nonvaccinated and noninfected; NVI, nonvaccinated and infected; VS, Silirum®vaccinated and noninfected; VSI, Silirum®vaccinated and infected; VH, HIMB vaccinated and noninfected; VHI, HIMB vaccinated and infected; StrSI, Map 316F Silirum®strain immunized and infected; StrHI, Mbv 1403 HIMB strain immunized and infected; AdjSI, MontanideTM Silirum® immunized and infected; AdjHI, MontanideTM HIMB adjuvant immunized and infected). Bars and vertical lines represent mean values and standard deviations, respectively. Significant differences determined by multiple comparisons were represented as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. FIGURE 4 | Kinetics of peripheral blood serum antibody levels. Results are expressed as S/P ratio of Map (A) and Mbv (B) specific antibodies of each vaccination group (n=5) (NV, nonvaccinated and noninfected; NVI, nonvaccinated and infected; VS, Silirum®vaccinated and noninfected; VSI, Silirum®vaccinated and infected; VH, HIMB vaccinated and noninfected; VHI, HIMB vaccinated and infected; StrSI, Map 316F Silirum®strain immunized and infected; StrHI, Mbv 1403 HIMB strain immunized and infected; AdjSI, MontanideTM Silirum®immunized and infected; AdjHI, MontanideTM HIMB adjuvant immunized and infected) and time sampled (dpv, days postvaccination). Vertical dotted red line represents the time of Map oral challenge (45 dpv). Significant differences determined by multiple comparisons were represented as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. significant differences were observed in VHI (Figure 4A). Additionally, no significant differences were observed in the Map antibody levels either between Silirum R and HIMB vaccination groups or between infected and noninfected vaccinated groups. Mbv-specific S/P values were significantly higher when comparing the VHI group with the VS, VSI, StrSI, StrHI, AdjSI, and AdjHI groups at 60, 90, 120, 150, and 190 dpv (Figure 4B). However, significant differences between VHI and VH were observed only at 120, 150, and 190 dpv (Figure 4B). Quantitative PCR and Bacteriology of Tissues and Feces Map detection was confirmed by qPCR (n=4) and bacteriological culture (n=3) within infected goats (n= 35). Animals positive to qPCR corresponded to (i) two goats from StrHI (mesenteric lymph node and the Peyer patches, respectively), (ii) one from VHI (Peyer patches), and (iii) one from AdjSI (Peyer patches). Besides, animals positive to bacteriological culture corresponded to one goat each from NVI, VHI (ileum, Peyer patches, and mesenteric lymph node Frontiers in Veterinary Science | www.frontiersin.org 8January 2022 | Volume 8 | Article 744568 Arteche-Villasol et al. Vaccination Against Caprine Paratuberculosis positives), and VSI (ileum and mesenteric lymph node positives). Only the Peyer patches from the goat from VHI showed positive results for both detection techniques. All tissue samples from noninfected groups were negative to IS900 sequence amplification by qPCR or bacteriological culture. Bacteriological culture of Map was negative in every fecal sample analyzed. Relative Peripheral Blood and Tissue Lymphocyte Subsets The mean and standard deviations of the relative proportions of three T lymphocytes (CD4+, CD8+, and CD3+), one γδ T lymphocyte, and two B lymphocyte (CD21+and CD20+) surface markers were estimated by flow cytometry. Along all groups, the highest relative proportion of positive cells in PBMCs isolated from whole blood corresponded to γδ T lymphocytes (30.46% ±3.55%), whereas the lowest proportion was observed in CD20+B lymphocytes (5.96 ±1.48%) throughout the study (Supplementary Table 1). However, despite statistical analysis showing significant oscillations (p <0.001) of the relative proportions of CD4+, CD8+, WC1+, CD21+, CD3+, and CD20+lymphocytes at different samplings, no significant differences were found between groups (p>0.05). Flow cytometry carried out in cells purified from tissue samples showed that the highest relative proportion levels corresponded to CD21+B lymphocytes in the ileum (39.47 ± 4.71%), Peyer patches (26.81 ±3.59%), and mesenteric lymph node (44.90 ±3.52%), whereas γδ T lymphocytes were the scarcest cell population in these samples (0.99 ±0.24%, 1.69 ±0.33%, and 0.92 ±0.26%, respectively). Statistical analysis showed a significant influence of vaccination and immunization with inactivated bacteria and adjuvants on the relative proportion of tissue lymphocyte subsets (p<0.05). However, results from comparisons between groups did not show a clear pattern between the relative proportion of lymphocyte subpopulations and the different groups likely on account of the individual variability observed within groups. Mean, standard deviation, and results of multiple comparisons of lymphocyte relative subpopulations in ileum, Peyer patches, and mesenteric lymph node are summarized in Supplementary Tables 2–4. Pathological Findings Gross lesions compatible with paratuberculosis were not found in any animal. Microscopic granulomatous lesions characteristic of Map infection were detected in all infected groups, with differences in the severity and distribution. The number of goats per group with lesions according to the tissue and the number of goats per group classified in terms of severity of the lesions are shown in Table 2. Goats with small and well-defined granulomas composed of macrophages, with an abundant pale cytoplasm and a large nucleus, escorted by a few lymphocytes located exclusively in the interfollicular area of the intestinal lymphoid tissue were classified as focal (n=5) (Figure 5A). Besides, goats with well-defined granulomas in the interfollicular area of the Peyer patches and also in the lamina propria closely associated with the intestinal lymphoid tissue were categorized as multifocal a(n=5) (Figure 5B). Finally, the presence of granulomatous lesions not only in the Peyer patches and related lamina propria but also in areas of the mucosa without any association with the lymphoid tissue was considered as multifocal b (n=13) (Figure 5C). No diffuse lesions were noticed in the tissues of any goat (n=0). VSI and VHI groups were the less affected with only one animal from each group developing granulomatous lesions, showing significant differences in comparison to NVI in which lesions were present in all animals (p<0.05). In addition, lesions from the VSI goat were classified as focal, whereas the goat from VHI group showed more severe lesions, categorized as multifocal b as all goats from the NVI. Besides, most of the goats immunized with strains (StrSI and StrHI) developed multifocal b forms, except for one animal from StrHI with a focal lesion. In contrast, among the animals immunized with adjuvants (AdjSI and AdjHI), only one animal from the AdjSI showed multifocal blesions, whereas focal and multifocal a lesions were found in TABLE 2 | Number of animals from each group with microscopic lesions consistent with paratuberculosis according to the examined tissue and its severity (focal, multifocal a,multifocal b, and diffuse). Group (n=5) Tissues Severity IL JJ JPP ICV MLN JLN ICLN Focal Multifocal a Multifocal b Diffuse Total NVI 3/5 2/5 5/5 3/5 1/5 1/5 — — — 5/5 — 5/5 VSI 1/5 — 1/5 — — — 1/5 1/5 — — — 1/5a* VHI 1/5 — 1/5 — — — — — — 1/5 — 1/5a* StrSI 3/5 2/5 3/5 1/5 3/5 1/5 1/5 — — 3/5 — 3/5 StrHI 1/5 — 4/5 1/5 1/5 1/5 — 1/5 — 3/5 — 4/5 AdjSI 1/5 — 3/5 1/5 — — — — 2/5 1/5 — 3/5 AdjHI 1/5 — 3/5 1/5 1/5 — — 3/5 1/5 — — 4/5 IL, Ileum; JJ, jejunum; JPP, jejunal Peyer patches; ICV, ileocecal valve; MLN, mesenteric lymph node; JLN, jejunal lymph node; ICLN, ileocecal lymph node; NVI, Nonvaccinated and infected; VSI, Silirum®vaccinated and infected; VHI, HIMB vaccinated and infected; StrSI, Map 316F Silirum®strain immunized and infected; StrHI, Mbv 1403 HIMB strain immunized and infected; AdjSI, MontanideTM Silirum®adjuvant immunized and infected; AdjHI, MontanideTM HIMB adjuvant immunized and infected. 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(2013) 43:881–4. doi: 10.1111/eci.12132 Conflict of Interest: 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. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2022 Arteche-Villasol, Gutiérrez-Expósito, Elguezabal, Sevilla, Vallejo, Espinosa, Ferreras, Benavides and Pérez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Veterinary Science | www.frontiersin.org 16 January 2022 | Volume 8 | Article 744568