Nonspecific protection of heat-inactivated Mycobacterium bovis against Salmonella Choleraesuis infection in pigs

Vaz‑Rodriguesetal. Veterinary Research (2022) 53:31 https://doi.org/10.1186/s13567‑022‑01047‑8 RESEARCH ARTICLE Nonspecific protection ofheat‑inactivated Mycobacterium bovis againstSalmonella Choleraesuis infection inpigs Rita Vaz‑Rodrigues1†, Elisa Ferreras‑Colino1†, María Ugarte‑Ruíz2, Michele Pesciaroli3, Jobin Thomas4,5, Teresa García‑Seco2, Iker A. Sevilla6, Marta Pérez‑Sancho2,7, Rafael Mateo1, Lucas Domínguez2,7, Christian Gortazar1* and María A. Risalde8,9,10 Abstract Trained immunity is the capacity of innate immune cells to produce an improved response against a secondary infec‑ tion after a previous unrelated infection. Salmonellosis represents a public health issue and affects the pig farming industry. In general, vaccination against salmonellosis is still facing problems regarding the control of distinct serovars. Therefore, we hypothesized that an immunostimulant based on heat inactivated Mycobacterium bovis (HIMB) could have an immune training effect in pigs challenged with Salmonella enterica serovar Choleraesuis (S. Choleraesuis) and decided to explore the amplitude of this non‑specific immune response. For this purpose, twenty‑four 10 days‑ old female piglets were randomly separated in three groups: immunized group (n = 10) received orally two doses of HIMB prior to the intratracheal S. Choleraesuis‑challenge, positive control group (n = 9) that was only challenged with S. Choleraesuis, and negative control group (n = 5) that was neither immunized nor infected. All individuals were necropsied 21 days post‑challenge. HIMB improved weight gain and reduced respiratory symptoms and pulmonary lesions caused by S. Choleraesuis in pigs. Pigs immunized with HIMB showed higher cytokine production, especially of serum TNFα and lung CCL28, an important mediator of mucosal trained immunity. Moreover, immunized pigs showed lower levels of the biomarker of lipid oxidation malondialdehyde and higher activity of the antioxidant enzyme superoxide dismutase than untreated challenged pigs. However, the excretion and tissue colonization of S. Choleraesuis remained unaffected. This proof‑of‑concept study suggests beneficial clinical, pathological, and heterol‑ ogous immunological effects against bacterial pathogens within the concept of trained immunity, opening avenues for further research. Keywords: Heat‑inactivated Mycobacterium bovis, pig, Salmonella Choleraesuis, trained immunity © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Introduction Traditional vaccination targets specific pathogens, relying on the adaptive immune response mediated by T and B lymphocytes [1, 2]. Nevertheless, a growing body of evidence suggests that the innate immune response is also enhanced after stimulation with certain antigens, partially protecting against infection with the same pathogen or a different one [3]. Here lies the concept of trained immunity that does not imply a high level of specificity nor amplification as it happens with adaptive immunity and which involves innate immune cells such as macrophages (MΦs) and natural killer (NK) cells [4, 5], as well as proinflammatory cytokines such as interleukin (IL)-1α and tumour necrosis factor α (TNFα) or Open Access *Correspondence: christian.gor[email protected] †Rita Vaz‑Rodrigues and Elisa Ferreras‑Colino contributed equally to this work 1 SaBio (Health and Biotechnology), Instituto de Investigación en Recursos Cinegéticos IREC (UCLM‑CSIC), Ciudad Real, Spain Full list of author information is available at the end of the article Page 2 of 16 Vaz‑Rodriguesetal. Veterinary Research (2022) 53:31 reactive oxygen species (ROS) [6] as mediators. It seems that trained immunity relies on epigenetic and metabolic reprogramming for enhancing the functional state of innate immune cells, cytokine production and sustaining cellular memory [7–9], which could be responsible for the nonspecific protective effects produced by some immunostimulants [10, 11]. One of the most used vaccines as a model to study trained immunity mechanisms is Bacillus Calmette-Guérin (BCG), due to its ability to induce nonspecific crossprotection against a vast number of pathogens. In murine models, heterologous BCG stimulation has shown to reduce the acquisition of infections caused by Candida albicans, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Schistosoma mansoni, Plasmodium sp. and Babesia sp. [6, 12, 13]. Moreover, various studies revealed that infant immunization with BCG reduced the incidence of acute lower respiratory infections [14, 15]. However, oral administration of this live attenuated vaccine led to BCG shedding and detection in lymphoid organs several days postimmunization [16, 17]. To prevent this, the use of inactivated immunostimulants seems promising, since it decreases the possibility of vaccine strain spreading and makes deployment logistics easier [18]. An immunostimulant based on heat-inactivated Mycobacterium bovis (HIMB) was developed in 2011 for use in pigs and wild boar (Sus scrofa) against tuberculosis, with similar results as oral BCG in the reduction of lesions and mycobacterial culture scores [19]. Studies using HIMB in different species and applying various routes of administration proved its homologous effect against Mycobacterium bovis (M. bovis) [20–23]. In this regard, it has been demonstrated that killed mycobacteria activate a training effect in the lytic phase of phagocytosis in MΦs [24], as well as an increase in pro-inflammatory cytokines and the complement component 3 (C3) in immunized animals [25–27]. The immunological mechanisms triggered by HIMB stimulation could also favour nonspecific protection against unrelated pathogens compatible with the concept of trained immunity. However, to the best of our knowledge, there are no studies assessing the heterologous protection capacity of inactivated mycobacteria, in other words, experiments using infectious agents other than mycobacteria for challenge. Salmonella enterica subspecies enterica serotype Choleraesuis (S. Choleraesuis), is a zoonotic pathogen that causes pneumonia and septicaemia in swine and can lead to miscarriage in sows [28, 29]. Aside from taking a toll on the pig farming industry, leading to major economic losses due to reductions in daily weight gain and therapy expenses [30], it also represents a threat to public health [31]. Furthermore, the prevalence of this serovar amongst its reservoir, the wild boar, is increasing in Europe [32, 33] with outbreaks in Italy [34] and Spain [35]. This serovar was also reported in Danish pig herds [36] and in weaned piglets from Serbia [37]. The increasingly common resistance to conventional antimicrobial agents and the limited capacity of vaccines to control distinct Salmonella serovars creates the need to explore trained immunity activators (immunostimulants) as an alternative or complementary control tool [38, 39]. The objective of this study was to evaluate if an immunostimulant based on heat inactivated M. bovis (HIMB) elicits a protective effect in pigs challenged with S. Choleraesuis. We explore the amplitude and efficacy of this non-specific immunological stimulation, a process that falls within the trained immunity concept. We hypothesize that HIMB stimulates the innate immune system and may protect pigs against S. Choleraesuis challenge. Materials andmethods Animals andexperimental design Twenty-four 10days-old Landrace x Large White hybrid female piglets with homogeneous weights were obtained from a pig production farm for experimental animals. They were in good health, free of clinical signs of enteric disease and seronegative to Salmonella species. Also, Salmonella spp. was not isolated from their mother’s stools. Seven days prior to start the study, they were housed in class III biocontainment animal facilities (BSL-3) situated at VISAVET Health Surveillance Centre (Madrid, Spain) for acclimatization. All animals received continuous access to water, nonmedicated pig feed and veterinary care. These individuals were randomly assigned to the experimental groups, identified via microchip and ear tag and housed in separated rooms as follows: the immunized group (n = 10) received two oral doses (with an interval of 3weeks) of HIMB prior to the intratracheal S. Choleraesuis-challenge, the positive control group (n = 9) that was only challenged with S. Choleraesuis, and the negative control group (n = 5) that was neither immunized nor infected. The challenge was done using an 18G needle after sedation with xylazine (Xilagesic 2%; Laboratories Calier, Barcelona, Spain). Animals were handled several days during the experiment for stool sample collection (SS), blood withdrawal (BW), body temperature (BT) and weight (W) measurements. They were also monitored for the appearance of clinical signs (CS) (Table1). The stool samples were processed by microbiological culture to evaluate faecal shedding. The whole blood collected served for determining the activity of glutathione peroxidase (GPx, EC 1.11.1.9) and superoxide dismutase (SOD, EC 1.15.1.1), while malondialdehyde (MDA) levels were analysed via plasma samples. The serum obtained was used for determining Page 3 of 16 Vaz‑Rodriguesetal. Veterinary Research (2022) 53:31 pig complement C3, cytokine (IL-1β, IL-10 and TNFα) concentration and antibody titers against Mycobacterium tuberculosis complex (MTC). All piglets were euthanized at 21 days post-infection (dpi) by captive bolt after sedation with xylazine (Xilagesic 2%, Laboratories Calier, Barcelona, Spain). Later, they were subjected to necropsy to assess the presence and extension of S. Choleraesuis macroscopic lesions in several organs (Table2). The evaluation of these findings was performed using a lesion score: absent = 0, mild = 1, moderate = 2 and severe = 3. We collected samples from the following tissues: palatine tonsil, tracheobronchial lymph nodes (LNs), spleen and lung (cranial and caudal lobes). Samples from the lymphoid organs (palatine tonsil, tracheobronchial LNs and spleen) were cultured to evaluate tissue colonization. Lung and tracheobronchial LNs samples were fixed in 10% neutral buffered formalin, embedded in paraffin wax and routinely processed for histopathology. Furthermore, other lung tissue samples were also stored at −80 °C with RNAlater Stabilization Solution (Thermo Scientific, Wilmington, USA) for molecular studies. HIMB immunostimulant The oral immunostimulant used consisted of 2 mL of sterile PBS containing approximately 107 heat inactivated CFU/mL of a M. bovis field isolate (strain 1403; spoligotype SB0339) that had been obtained from a naturally infected wild boar. The vaccine preparation followed the protocol described by Garrido etal. [19], apart from an extended inactivation step at 83°C for 45min. Bacterial concentration of HIMB was determined prior to inactivation by measuring the turbidity of the suspension in MacFarland scale using a VITEK® DensiCHEK® (BioMerieux) and by plating a serially diluted aliquot onto agar-solidified Middlebrook 7H9 with glycerol (0.2% v/v) and OADC (10% v/v) (Becton Dickinson, Franklin Lakes, NJ, USA). Challenge The S. Choleraesuis variant Kunzendorf used as challenge strain (DICM15/00069, VISAVET reference) was isolated from the spleen of a naturally infected wild boar and cultivated in a Luria–Bertani broth at 37°C for 24h, following the protocol described by Ibrahim etal. [40]. Each dose consisted of 106CFU S. Choleraesuis in one mL. Microbiological culture andSalmonella excretion To analyse faecal shedding and bacteria tissue distribution the semiquantitative microbial culture method based on ISO 6579:2002/AMD 1:2007 was applied but by performing ten-fold serial dilutions to enable the estimation of bacterial concentrations in each sample [41]. Once known the last dilution where bacterial growth occurs, the number of CFU/gram per sample was calculated. Histopathology A standard procedure for haematoxylin and eosin (HE) staining was used for microscopical examination of lung and tracheobronchial LNs. Although tissues of all animals were processed for histopathology, only samples from immunized and control positive groups were analysed. The main lesions caused by S. Choleraesuis in these organs [42–44], were evaluated giving a lesion score based on the severity and distribution of the lesions in each individual, where absent = 0, mild = 1, moderate = 2 and severe = 3. The histopathological lesions considered Table 1 Data collection calendar indicating the days each measure or monitorization was made Days post‑infection (dpi) that animals were handled for measurement of body temperature (BT), weight (W), blood withdrawal (BW), collection of stool samples (SS) and clinical sign (CS) monitoring. Days Salmonella Choleraesuis post-infection (dpi) 0 1 2 3 7 9 14 15 20 21 BT ✔✔✔✔✔ ✔✔✔✔ W✔ ✔ ✔ ✔ BW ✔ ✔ ✔ ✔ ✔ SS ✔ ✔ ✔ ✔ CS ✔ ✔ ✔ ✔ ✔ ✔ Table 2 Macroscopic lesions per organ taken into account for scoring Organ Macroscopic lesions Lymph nodes Lymphadenomegaly and congestion Lung Interstitial pneumonia Heart Hydropericardium Liver Focal necrosis Spleen Splenomegaly and hyperaemia Small intestine Enteritis, congestion and petechiae Kidney Focal necrosis and petechiae Page 4 of 16 Vaz‑Rodriguesetal. Veterinary Research (2022) 53:31 for lung scoring were oedema (alveolar or interstitial), congestion, haemorrhages, hemosiderosis, inflammatory infiltrate (mononuclear, polymorphonuclear [PMN] or mixed), bronchial exudate, hyperplasia of seromucous glands and of epithelial cells, and necrosis. As for the tracheobronchial LNs the following lesions were considered: oedema, congestion, haemorrhages, hemosiderosis, lymphoid depletion, and necrosis. Oxidative stress biomarkers (MDA, GPx andSOD) MDA plasma levels were measured using high-performance liquid chromatography (HPLC), as previously described by Agarwal and Chase [45]. Concentrations were expressed as mmol MDA/L of plasma samples and estimated by applying the linear regression equation of the standard curve to the unknown sample peak-area. The activities of GPx and SOD were determined spectrophotometrically using the Ransel and Ransod kits (Randox Laboratories, Crumlin, UK), respectively, following the instructions from the manufacturer. The results of the GPx and SOD activity were expressed in µmol/L of whole blood. Analysis ofcomplement C3 inserum Pig serum C3 concentration was measured using a commercial sandwich ELISA kit (Cusabio Technology LLC, Houston, USA), following the manufacturer’s guidelines. A standard curve was generated alongside with a regression analysis to determine each sample C3 concentration in µg/mL. Serum cytokines (IL-1β, IL-10 andTNFα) The serum concentrations of the proinflammatory cytokines IL-1β, IL-10 and TNFα were examined via specific swine commercial sandwich ELISA Kits (Invitrogen, Waltham, USA), following the instructions from the manufacturer. A standard curve was run per assay, allowing the determination of each cytokine concentration in pg/mL. Antibody titers againstMTC Serum samples were analyzed by ELISA to detect antibodies against MTC in all the groups studied at the different blood collection time points. Bovine tuberculin purified protein derivative (bPPD) (CZ Veterinaria SA, Porriño, Spain) and an immunopurified subcomplex protein from bPPD, named P22 [46], were used as antigens in an indirect in-house ELISA previously described by Thomas etal. [47]. The estimated sensitivity and specificity of this ELISA in swine was 77.3% and 97.3% for PPDb, as well as 84.1% and 98.4% for P22, respectively [47]. Assay ofpulmonary cytokines byreal time qPCR Lung tissue samples were used to extract mRNA. Firstly, 15mg of tissue was disrupted with a scalpel and homogenized using a needle and syringe. Secondly, total mRNA was isolated using Rneasy Plus Mini Kit (Qiagen, Hilden, Germany), following manufacturer’s instructions. The reverse transcription of total mRNA into cDNA was performed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, USA), following the guidelines of the manufacturer. We used a total volume reaction of 20 µL containing: 2 µL of sample mRNA, 1 µL of iScript Reverse Transcriptase, 4 µL of 5× iScript Reaction Mix and 13 µL of nuclease-free water. The complete reaction mix was incubated in a thermal cycler for priming during 5min at 25°C, then the reverse transcription was carried out for 20min at 46°C followed by inactivation at 95°C for 1min. Both concentration (ng/µL) and purity of mRNA and cDNA samples were assessed by quantification of the nucleic acids at an optical density of 260nm (OD260) and the ratio of absorbance at 260/280 nm, using a Nanodrop One spectrophotometer (Thermo Scientific, Waltham, USA). At the end, concentrations were standardized at 20ng/µL for mRNA and 200ng/µL for cDNA. Afterwards, all samples were stored at −80°C. The amplification of the synthesized cDNA was realized using the CFX96 real-time PCR detection system (Bio-Rad, Hercules, USA) and the quantification was performed by utilizing SYBR green chemistry (Power SYBR Green, Applied Biosystems, Waltham, USA). All real time PCR reactions were carried through in a 96 well plates (Applied Biosystems, Waltham, USA). The concentration selected for primer working solution was 10μM. For a total volume of 20 µL, the PCR mixture contained: 10 µL of SYBR Green Master Mix (Bio-Rad, Applied Biosystems, Waltham, USA), 1 µL of primer forward, 1 µL of primer reverse, 2 µL of sample cDNA and 6 µL nucleasefree water. For each PCR reaction, every sample had two technical replicates and two negative controls. Cyclophilin was the housekeeping gene that was used to normalize the expression of the cytokines analysed, applying the 2−ΔΔCt method (relative quantification). Table3 lists the sequence of the forward and reverse primers (SigmaAldrich, Darmstadt, Germany) used alongside with its amplicon length. Table4 shows the distinct thermal cycle parameters applied for each cytokine analysed, PCR efficiency and R values obtained. Statistical analysis Data analysis was performed in R 4.0.4 using a fit linear mixed-effects model for data collected ante-mortem (clinical signs, temperature increment, body weight Page 5 of 16 Vaz‑Rodriguesetal. Veterinary Research (2022) 53:31 gain, C3, serum cytokines, oxidative stress biomarkers and faecal shedding) and the non-parametric test Mann–Whitney–Wilcoxon for post-mortem data (bacterial tissue colonization, macroscopic and histopathological lesions and cytokine expression in the lung). To reduce skewness of the original data, a logarithmic transformation was applied to faecal shedding and bacterial tissue colonization. For the fit linear mixedeffects model [outcome variable ~ group * day + (1 | individual)] each ante-mortem data was used as the outcome variable, fixed factors were “group” and “day” (categorical variable), and “individual” was included as a random factor. The estimation method used in this case was the Restricted Maximum Likelihood (REML) and, to prove model fit, the residual plot was analysed to ensure lack of patterns and normality presence. As for the post-mortem data, only two groups were statistically analysed as the control group served as a reference of the basal level and was not included in this statistical analysis. Correlation between cytokine expression and microscopic lung lesion score was executed via Spearman’s correlation test due to the non-gaussian data distribution used. The data are shown as mean ± standard error of the mean (SE). For all analysis, statistical significance was declared using a p-value ≤ 0.05 with a confidence level (CL) of 95%. Results Oral immunization withHIMB improves weight gain andreduces S. Choleraesuis clinical signs inpigs Clinical signs were mainly respiratory (dyspnoea, cough, sneezing and aphonia) and only individuals from the positive control group presented severe breathing difficulty. Digestive symptoms (diarrhoea) were mild and sporadic in both groups. Apathy and depression were also registered, mostly within the positive control individuals. Only one animal, belonging to the immunized group, had to be euthanized 13 dpi due to septicaemia. During this experiment, a total of 7/9 (77.8%) individuals from the positive control group were symptomatic versus 4/10 (40.0%) of animals from the immunized group. Piglets from the negative control did not show signs of illness. Although non-significant (p > 0.05), the mean clinical signs score was higher in the positive control group and appeared earlier than in the immunized group, emphasizing the rapid onset of this disease (Figure1A). Moreover, respiratory signs were considerably more pronounced in positive controls, with a significant difference at 7 dpi (p = 0.0003) (Figure1B). Body temperature increment was evident for the positive control group since 1 dpi. Meanwhile, the immunized group maintained its temperature constant, with a slight increase at 7 and 14 dpi. Statistical analysis showed Table 3 Details of primer sequences for RT-PCR assessment of pulmonary cytokines in pigs challenged with S. Choleraesuis CCL28, C–C Motif chemokine ligand 28; IFNγ, Interferon gamma; IL‑1α, Interleukin 1 alfa; IL‑8, Interleukin 8; TNFα, Tumour necrosis factor alpha. Target Amplicon size (base pairs) Primer Forward (5ʹ–3ʹ) Primer Reverse (5ʹ–3ʹ) References TNFα 134 ACT CGG AAC CTC ATG GAC AG AGG GGT GAG TCA GTG TGA CC [76] IFNγ 79 TGG TAG CTC TGG GAA ACT GAATG GGC TTT GCG CTG GAT CTG [76] CCL28 145 GCT GCT GCA CTG AGG TTT C TGA GGG CTG ACA CAG ATT C [68] IL-1α 274 GCT GCT GTG CTA AAT AAC CT CTT GTG GCA ATA AAC AAC TTT [77] IL-8 240 TCT TGG CAG TTT TCC TGC TT CAA CCT TCT GCA CCC ACT TT [77] Cyclophilin 369 TAA CCC CAC CGT CTT CTT TGC CAT CCA ACC ACT CAG [69] Table 4 Quantitative PCR conditions and efficiency, R values and number of cycles used for each porcine cytokine and cyclophilin cDNA CCL28, C–C Motif chemokine ligand 28; IFNγ, Interferon gamma; IL‑1α, Interleukin 1 alfa; IL‑8, Interleukin 8; TNFα, Tumour necrosis factor alpha. PCR conditions and efficiency Cytokine Initial Denaturation Denaturation Annealing Elongation Number of PCR cycles PCR efficiency R TNFα 95 °C (10 min) 95 °C (15 s) 60 °C (1 min) 40 2.168 0.994 IFNγ 95 °C (10 min) 95 °C (15 s) 60 °C (1 min) 40 1.982 0.999 CCL28 95 °C (3 min) 95 °C (15 s) 56 °C (30 s) 60 °C (30 s) 45 2.097 0.997 IL‑1α 94 °C (2 min) 94 °C (20 s) 60 °C (1 min) 45 1.983 0.999 IL‑8 94 °C (2 min) 94 °C (20 s) 60 °C (1 min) 45 1.992 0.998 Cyclophilin 94 °C (3 min) 94 °C (45 s) 54 °C (45 s) 60 °C (45 s) 30 1.864 0.999 Page 6 of 16 Vaz‑Rodriguesetal. Veterinary Research (2022) 53:31 significantly higher temperatures of positive control individuals when compared to immunized ones (p = 0.0076) and negative controls (p = 0.0125) (Figure1C). No significant differences were registered between the negative control and the immunized group (p > 0.05) (Figure1D). There were statistically significant differences in daily weight gain between groups (group*day interaction, p = 0.0008). Weight gain was significantly smaller in the positive control group as compared to the negative control (p = 0.0041) and immunized (p = 0.0441) groups (Figure1E). A strong effect of group and day variables was evident on weight gain, being higher for negative control and immunized groups (Figure1F). Figure1 Clinical signs, temperature increment and weight gain in pigs infected and non-infected with Salmonella Choleraesuis. Evolution of clinical signs score (A), temperature increment (C) and body weight gain (E) per group and days post‑infection. Main effect plot for respiratory signs (B), temperature increment (D) and weight gain (F) using the combined effect of fixed variables group and day. CN: negative control group; CP: positive control group; I: immunized group; SE: standard error of the mean. Page 7 of 16 Vaz‑Rodriguesetal. Veterinary Research (2022) 53:31 HIMB isnotcapable ofreducing S. Choleraesuis shedding ororgan dissemination S. Choleraesuis shedding peaked at 14 dpi for the positive control and immunized groups and was also recorded at 21 dpi but with less CFU/gram of faeces (data not shown). Differences in bacterial faecal excretion between groups were non-significant (p > 0.05). Bacterial colonization was especially evident in the tracheobronchial LNs, affecting positive control and immunized groups equally (around 70% of all animals). Bacteria were isolated from palatine tonsils of 66% of the positive control individuals, contrasting with only 24% of the immunized ones. Spleen was the less colonized organ, with three animals (33%) from the positive control group affected and only one individual (12.5%) from the immunized group. Despite these observations, there were no significant differences (p > 0.05) between groups for total tissue or individual organ colonization (palatine tonsil, spleen and tracheobronchial LNs). However, a reduction of almost 102 CFU/g between immunized and positive control groups for total tissue bacterial colonization was observed (data not shown). Immunostimulation withHIMB reduces S. Choleraesuis gross pulmonary lesion score inpigs After S. Choleraesuis challenge, post-mortem examinations revealed that macroscopic lesions were present across 8/9 (88.9%) of individuals from positive control and immunized groups. Control negative animals showed no remarkable gross lesions. The main lesions observed in positive control and immunized groups were interstitial pneumonia, mostly located in the cranial and middle lobes, combined with areas of atelectasis (Figure2A). In this case, 7/9 (77.8%) of pigs from the positive control group presented gross pulmonary lesions versus 4/9 (44.4%) from the immunized group (Figure3A). Regarding lesion severity, 44% of the positive control group individuals presented four affected lobes with interstitial pneumonia, while in immunized individuals there were only one (25%) or two lobes (12.5%) with these lesions (Figures2Band 3A). Furthermore, the lung was one of the most affected organs. Analysing individual pulmonary macroscopic lesions demonstrated that lung lesion scores were higher in the positive control group (p = 0.0472) (Figure3B). The presence of lesions in the tracheobronchial LNs was observed in 38% of the positive control animals, compared to 75% of the immunized individuals. LNs were mostly hypertrophic and congestive in both groups. Hydropericarditis was registered in 38% of the positive control animals and in 13% of those from the immunized group. Regarding intestinal lesions, 25% of the positive control animals showed enteritis and, as for the immunized group, 38% only presented intestinal vascular changes (congestion or petechial haemorrhages). No signs of enteritis were observed in the immunized group (Figure2B). Splenomegaly was reported in 25% of the positive control group and 13% of the immunized one. Moreover, one individual from Figure2 Macroscopic lesions in pigs infected with Salmonella Choleraesuis at 21days post-infection. A Lung of an animal from the positive control group presenting an increased size and moderate interstitial pneumonia in the right cranial and middle lobes (white asterisk). B Thoracic and abdominal cavities of an animal from the immunized group presenting organs without lesions. C Spleen and intestine of an animal from the positive control group showing adherence of connective tissue (black asterisks); moreover, the spleen presents multiple abscesses of purulent material (inset). Page 8 of 16 Vaz‑Rodriguesetal. Veterinary Research (2022) 53:31 the positive control group presented seven encapsulated splenic abscesses with purulent content (Figure2C). Lastly, multifocal necrotic hepatic lesions were observed in two of the immunized animals and three liver abscesses were present in one individual from the positive control group. The analysis of the total macroscopic lesions score revealed no significant differences between immunized and positive control groups (p > 0.05), although the total mean score of the immunized group (3.38 ± 0.86) was 42% smaller than the positive control (5.89 ± 0.98) one. Oral immunization withHIMB reduces S. Choleraesuis histopathological thoracic lesion score inpigs For both challenged groups, the main microscopic lesions observed in lung tissue were mononuclear (MΦs and lymphocytes) or mixed (MΦs, lymphocytes, and neutrophils) infiltrates, bronchial epithelial shedding, congestion, interstitial oedema, interstitial haemorrhage and hemosiderosis. Moderate-severe mononuclear infiltrate, mainly constituted by MΦs, was present in 44% of the positive control animals (Figure 4A), while 77% of the individuals from the immunized group presented a mild mononuclear infiltrate (Figure4B). Only in the positive control group, 44% of individuals presented a mild/moderate alveolar oedema. Liquefactive necrosis was found in two animals from the positive control group and in one individual from the immunized group. The total microscopic lung lesion score was significantly higher (p = 0.0204) in the positive control group when compared to the immunized one (Figure5A). Tracheobronchial LNs from 75% of the positive control group revealed moderate lymphoid depletion, whilst only one individual from the immunized group had a mild presentation of this lesion (Figures4Cand D). Also, congestion was found in more individuals from the positive control group (57%) than the immunized one (25%). Oedema occurred in two individuals from both groups, while hemosiderosis and liquefactive necrosis were only found in one individual from the immunized group. Differences in total tracheobronchial LN scores between groups were marginally significant (p = 0.0639) (Figure5B), showing a tendency for lesion reduction in the immunized group. The total thoracic microscopic lesions score (sum of lung and tracheobronchial LNs lesion score) was larger (p = 0.0334) in the positive control group when compared to the immunized one (Figure5C). HIMB stimulation lowers MDA levels andenhances SOD activity inpigs infected withS. Choleraesuis Plasma MDA variation levels revealed a significant concentration peak 1 dpi for the positive control group compared to the immunized group (p = 0.0444) (Figure6A). This led to a negative slope on the main effect plot for the positive control group (Figure 6B), uncovering the impact that factor group combined with day had on this parameter. By contrast, SOD activity variation of immunized piglets showed an increasing trend from 0 to 1 dpi, maintaining higher values than the other groups until 7 dpi (p > 0.05) (Figure6C). As a result, the positive slope Figure3 Characterization of lung macroscopic lesions caused by Salmonella Choleraesuis at 21days post-infection. A Lung gross lesions severity and percentage of piglets affected in each group. B Lung macroscopic lesions score separated per groups. CP: positive control group; I: immunized group; SE: standard error of the mean; Type I: interstitial pneumonia with 1 lobe affected; Type II: interstitial pneumonia with 2 lobes affected; Type III: interstitial pneumonia with 3 lobes affected; Type IV: interstitial pneumonia with 4 lobes affected. Page 9 of 16 Vaz‑Rodriguesetal. Veterinary Research (2022) 53:31 Figure4 Histopathological lesions in pigs infected with Salmonella Choleraesuis at 21days post-infection. A Lung of an animal from the positive control group presenting a moderate mixed interstitial inflammatory infiltrate (*), mainly composed of macrophages (MΦs) and lymphocytes (inset). B Lung of an animal from the immunized group showing a light thickening of the septa (*), due to infiltrate particularly of MΦs (inset). C Tracheobronchial lymphoid node of an animal from the positive control group presenting severe lymphoid depletion and compared with an animal from the immunized group (D). Haematoxylin–eosin stain. Figure5 Microscopic lesions score in pigs infected with Salmonella Choleraesuis. Lung (A), tracheobronchial lymph nodes (B) and total thoracic (C) microscopic lesions score in pigs infected with Salmonella Choleraesuis: a comparison between immunized and positive control groups. CP: positive control; I: immunized; LNs: lymph nodes; NS: not significant; TB: tracheobronchial. Page 16 of 16 Vaz‑Rodriguesetal. Veterinary Research (2022) 53:31 • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? 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