Breed-specific immune tolerance to Leishmania infantum in a native Mediterranean canine population

Breed-specific immune tolerance to Leishmania infantum in a native Mediterranean canine population | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Breed-specific immune tolerance to Leishmania infantum in a native Mediterranean canine population Lola Martínez-Sáez, Pablo Jesús Marín-García, Raffaella Cocco, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8367154/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Mar, 2026 Read the published version in Parasites & Vectors → Version 1 posted 9 You are reading this latest preprint version Abstract Canine leishmaniosis caused by Leishmania infantum remains a major zoonotic concern in the Mediterranean basin, where native breeds may have evolved adaptive immune mechanisms against infection. This study evaluated cytokine and growth factor profiles in Fonni dogs, a Sardinian native breed, and German Shepherds exposed to L. infantum and other vector-borne pathogens. Samples were collected as part of routine clinical practice, and no procedures were performed for research purposes. Fifty-nine clinically healthy dogs were analyzed for seropositivity to Anaplasma phagocytophilum , Ehrlichia canis , Leptospira spp., Leishmania infantum , and Rickettsia spp., and for serum levels of eleven cytokines and growth factors using a multiplex immunoassay. Fonni dogs showed significantly higher serum levels of IL-10, NGF-β, IFN-γ, TNF-α, and VEGF-α compared with German Shepherds. L. infantum and Rickettsia spp. positive animals exhibited elevated IL-10, NGF-β, and TNF-α concentrations. Age influenced several cytokines, with young Fonni dogs displaying the highest IL-10 and NGF-β values, while TNF-α and MCP-1 increased with age. Correlation and principal component analyses revealed distinct clustering between breeds, highlighting coordinated regulation of pro-inflammatory and angiogenic mediators (IL-6, TNF-α, MCP-1, VEGF-α). The elevated values of several cytokines in Fonni dogs suggest a breed-specific immune tolerance-like phenotype consistent with reduced immunopathology under endemic exposure. These data support a breed-associated regulatory/pro-inflammatory immune balance in Fonni dogs, consistent with a tolerance-like immune phenotype under endemic exposure. cytokines Fonni dog immune tolerance Leishmania Mediterranean breeds Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Vector-borne diseases (VBDs) are among the most significant infectious threats to domestic dogs worldwide, particularly in the Mediterranean basin, where climatic conditions favor the proliferation of phlebotomine sandflies, ticks, and fleas that transmit protozoal, bacterial, and rickettsial agents. Among these pathogens, Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis [ 1 ], a disease endemic to Africa, Asia, the Americas, and Europe [ 2 ]. Within Europe, leishmaniasis persists in the Mediterranean region, particularly in Spain and Italy [ 3 , 4 ], where the domestic dog ( Canis lupus familiaris ) serves as the primary reservoir host [ 5 ]. However, dogs in these regions are often exposed not only to L. infantum , but also to other vector-borne pathogens such as Anaplasma phagocytophilum , Ehrlichia canis , Leptospira spp., and Rickettsia spp., which may circulate simultaneously in overlapping ecological niches [ 6 – 9 ]. Co-infections or sequential exposures to multiple VBDs can modulate host immunity, potentially affecting both susceptibility to L. infantum and the clinical outcome of infection [ 10 , 11 ]. The immunopathogenesis of canine leishmaniosis depends largely on the host’s ability to mount a protective Th1 immune response, characterized by IFN-γ, IL-2, and IL-12 production, while disease progression is often associated with elevated IL-10 and other cytokines related to anti-inflammatory and angiogenic activity [ 12 – 15 ]. Interestingly, some Mediterranean dog breeds show a natural resistance to L. infantum infection. For instance, the Ibizan hound and Cirneco dell’Etna display enhanced pro-inflammatory cytokine profiles and genetic polymorphisms in immune-related genes such as IFNG and IL6R , which appear to confer partial protection against infection [ 16 , 17 ]. This genetic and immunological diversity suggests that host factors play a critical role in disease progression. Nonetheless, little is known about other native Mediterranean breeds, including the Fonni’s dogs ( Cane Fonnese ), a rustic Sardinian molossoid breed recognized by the Italian Kennel Club (ENCI). Archaeological evidence suggests its origin dates to the Bronze Age, highlighting its long-standing adaptation to the Mediterranean pathogens [ 18 , 19 ]. Sardinia represents a unique epidemiological setting, where L. infantum coexists with a high prevalence of other vector-borne pathogens [ 20 , 21 ]. In such ecosystems, assessing the immune responses of local breeds may offer valuable insights into the mechanisms underlying natural resistance to infection. In this context, we investigated whether a native Mediterranean canine population chronically exposed to multiple vector-borne pathogens displays an immune profile consistent with tolerance to Leishmania infantum , using the Fonni dog as a naturally adapted host and the German Shepherds as a comparative breed. Material and methods This study is reported in accordance with the ARRIVE guidelines for animal research, including detailed descriptions of animal characteristics, inclusion and exclusion criteria, housing conditions, and statistical methods. Study population and sampling A cross-sectional observational study was conducted between September 2024 and April 2025 in Sardinia (Italy), a Mediterranean island endemic for L. infantum and other canine vector-borne pathogens. Fifty-nine privately owned, clinically healthy domestic dogs were included. Dogs were recruited consecutively during routine veterinary clinical visits, and the sample size corresponded to all animals meeting the inclusion criteria during the study period; therefore, no formal a priori sample size calculation was performed. Inclusion criteria comprised the absence of clinical signs compatible with canine leishmaniosis or other systemic infectious disease at the time of sampling, and owner consent to participate. Dogs showing overt clinical illness were excluded. All owners provided written informed consent before inclusion. Epidemiological data recorded for each animal included sex (male/female), age, and breed. Dogs were grouped by age into four categories: puppies (2–12 months), young (1–2 years), adults (3–8 years), and elder (9–11 years). Only dogs with documented pedigree certification were included in the breed analysis, which comprised the Fonni dog ( Cane Fonnese ) and German Shepherds. Dogs were maintained under typical household conditions according to owner routines, with ad libitum access to food and water. Information on antiparasitic prophylaxis was obtained from owners and clinical records. None of the enrolled dogs had received topical pyrethroid-based repellents or insecticidal collars in the 12 months preceding sampling. No animal had received immunosuppressive therapy during this period. The absence of standardized vector control measures was considered representative of natural exposure conditions in the study area. All samples were obtained as part of routine clinical practice, and no procedures were performed specifically for research purposes. Blood collection was carried out by licensed veterinarians, required less than three minutes per animal, and involved gentle manual restraint to minimize stress. All samples and associated data were anonymized before analysis. According to institutional and national regulations, and in line with European Directive 2010/63/EU on the protection of animals used for scientific purposes, formal ethical committee approval was not required for the use of surplus clinical samples not affecting patient management. For each dog, ten milliliters of blood were obtained by jugular venipuncture and divided into two tubes, one with EDTA to perform DNA extraction, and another without anticoagulant for serum separation. The tubes were maintained at room temperature. Serum samples were obtained by centrifuging the non-anticoagulated blood at 3000 rpm for 10 minutes. The separated serum was transferred to cryotubes and stored at -80ºC until infection and cytokine determination. Infection determination Serum samples were analyzed to detect antibodies against A. phagocytophilum , E. canis , L. infantum , Leptospira spp., and Rickettsia spp. Antibody detection was performed using the indirect immunofluorescence assay (IFAT) following the specific protocols for each pathogen. For A. phagocytophilum , slides containing the corresponding antigen (Fuller Laboratories) were used, and titers greater than 1:40 were considered positive. Detection of anti- E. canis antibodies followed the protocol described by [ 22 ]. The presence of antibodies against Leptospira spp. was determined using the Microscopic Agglutination Test (MAT), which is considered the reference serological method for diagnosing leptospirosis. The test was performed according to the standard procedures recommended by the World Organization for Animal Health (OIE). A panel of live Leptospira reference serovars representing the main pathogenic serogroups circulating in the region was used as antigens. Serum samples were initially diluted 1:50 in phosphate-buffered saline (PBS) and subsequently mixed with an equal volume of each live antigen suspension. After incubation at 28–30ºC for 1 hour, the mixtures were examined under a dark-field microscope to assess agglutination. A sample was considered positive when ≥ 50% of the leptospires were agglutinated compared to the control suspension. Positive sera were further titrated through serial two-fold dilutions to determine the highest dilution showing ≥ 50% agglutination, which was recorded as the antibody titer. Titers ≥ 1:100 were considered positive. Finally, anti- L. infantum IgG antibodies were detected using an in-house IFA in accordance with the World Organisation for Animal Health (WOAH, formerly OIE). Titers ≥ 1:80 were considered positive for both pathogens, and anti- Rickettsia spp. IgG antibodies were detected using a commercial canine Rickettsia spp. IgG Indirect Fluorescent Antibody Test (IFAT) kit, applying a positivity threshold of ≥ 1:128, following the manufacturer’s instructions. Cytokine quantification Serum levels of IL-2, IL-6, IL-8 (CXCL8), IL-10, IL-12/IL-23p40, NGF-β, IFN-γ, TNF-α, MCP-1 (CCL2), VEGF-α and SCF were quantified using the ProcartaPlex™ Canine Cytokine/Chemokine/Growth Factor Panel (ThermoFisher, Scientific, Waltham, MA, USA). Analyses were performed using the Luminex™ 200X detection system in serum samples, following the manufacturer's instructions. All samples were analyzed in duplicate, and intra-assay and inter-assay coefficients of variation were < 10% and < 15%, respectively. Cytokine values below the detection limit were replaced with half of the minimum detectable concentration. Statistical analysis Statistical analyses were performed using SAS software (version 9.2, North Carolina State University, USA). Cytokine concentrations were log10-transformed before analysis to reduce skewness. Linear mixed-effects models (PROC MIXED, REML estimation) were applied to evaluate the effects of breed, age group, sex, and serological status for each pathogen as fixed effects. Pairwise interactions between breed and age, and between breed and infection status, were included when biologically relevant. No random effects were specified, as each dog contributed a single observation. Model assumptions were assessed through inspection of residuals. P -values were adjusted for multiple testing across cytokines using the Benjamini-Hochberg false discovery rate (FDR), with adjusted q -values < 0.05 considered statistically significant. Descriptive statistics (mean ± SD, median, range) were calculated for all cytokines. Correlations among cytokine concentrations were evaluated using Spearman’s rank correlation coefficient (ρ), and correlation matrices were visualized as color-coded heatmaps with labelled p -values. Principal component analysis (PCA) was performed using MetaboAnalyst on autoscaled cytokine concentrations only. Categorical variables (breed, age group, infection status) were used exclusively for sample annotation and visualization and were not included in the PCA computation. to assess the quality of the dataset, identify potential outliers, and evaluate differences among the experimental groups. Before any analysis, all datasets were preprocessed using autoscaling, followed by normalization to the median [ 23 ]. Results Of the 59 dogs included in the study, fifteen were males (25.4%) and forty-four were females (74.6%). According to age, 8 dogs were classified as puppies (13.6%), 12 as young (20.3%), 26 as adults (44.1%), and 13 as elders (22.0%). Twenty-seven dogs belonged to the Fonni breed (45.8%), and thirty-two were German Shepherd (54.2%) (Table 1 ). Serological screening revealed antibodies against Rickettsia spp. in 21 dogs (35.6%), Leptospira spp. in 6 dogs (10.2%), E. canis in 3 dogs (5.1%), and L. infantum in 3 dogs (5.1%). No dogs were seropositive for A. phagocytophilum (Table 1 ). Serum cytokine and growth factor concentrations exhibited wide inter-individual variability. Median values were lowest for IFN-γ and highest for IL-8 and IL-12 (Table 2 ). After log10 transformation, residual inspection indicated acceptable model fit for all analytes. Table 1 Epidemiological data of dogs are included in the study. Variable Categories No. of dogs (%) Sex Male 15 (25.4%) Female 44 (74.6%) Age Puppy (2–12 months) 8 (13.6%) Young (1–2 years) 12 (20.3%) Adults (3–8 years) 26 (44.1%) Elder (9–11 years) 13 (22.0%) Breed Fonni 27 (45.8%) German Shepherd 32 (54.2%) A. phagocytophilum Positive 0 (0%) Negative 59 (100%) E. canis Positive 3 (5.1%) Negative 56 (94.9%) Leptospira spp. Positive 6 (10.2%) Negative 53 (89.8%) L. infantum Positive 3 (5.1%) Negative 56 (94.9%) Rickettsia spp. Positive 21 (35.6%) Negative 38 (64.4%) Table 2 Mean and standard deviation of all cytokines analyzed. IL: interleukin; NGF: nerve growth factor; IFN: interferon; TNF: tumor necrosis factor; MCP: monocyte chemoattractant protein; VEGF: vascular endothelial growth factor; SCF: stem cell factor. Cytokine serum levels are shown as mean ± standard deviation (SD). IL-2 (pg/mL) IL-6 (pg/mL) IL-8 (pg/mL) IL-10 (pg/mL) IL-12 (pg/mL) NGF-β (pg/mL) IFN-γ (pg/mL) TNF-α (pg/mL) MCP-1 (pg/mL) VEGF-α (pg/mL) SCF (pg/mL) Mean 146.7 299.7 1200.9 53.6 1197.6 79.3 7.7 33.2 56.5 26.5 201.1 Standard deviation 313.7 743.7 752.8 90.4 2421.5 180.7 11.6 93.7 59.7 21.4 326.5 Regarding fixed effects, no statistical differences were found between males and females; however, age exerted a significant effect on selected mediators (Table 3 ). IL-10 and NGF-β concentrations were highest in young dogs, whereas MCP-1 increased progressively with age, reaching the highest values in adult and elder animals ( q < 0.05, FDR-adjusted). VEGF-α also varied significantly across age groups, with higher concentrations in young and elder dogs compared with puppies. Marked breed-associated differences were observed (Table 3 ). Fonni dogs exhibited significantly higher serum concentrations of IL-10 (99.1 ± 115.3 pg/mL), NGF-β (146.2 ± 227.4 pg/mL), IFN-γ (11.2 ± 13.7 pg/mL), TNF-α (49.4 ± 94.3 pg/mL) and VEGF-α (37.3 ± 26.2 pg/mL) than German Shepherd dogs (15.1 ± 28.4, 22.8 ± 102.4, 4.8 ± 8.8, 19.5 ± 92.4 pg/mL and 17.4 ± 9.9 pg/mL, respectively). No breed-related differences were detected for IL-2, IL-6, IL-8, IL-12, MCP-1, or SCF. Table 3 Cytokine serum levels according to the variables. Bold numbers show statistical differences ( q < 0.05, Benjamini-Hochberg FDR-adjusted) in cytokine serum levels. Different superscripts in the same row indicate statistical differences. P -values correspond to linear mixed-effects models on log-transformed data with Benjamini-Hochberg FDR correction. IL: interleukin; NGF: nerve growth factor; IFN: interferon; TNF: tumor necrosis factor; MCP: monocyte chemoattractant protein; VEGF: vascular endothelial growth factor; SCF: stem cell factor. Cytokine serum levels are shown as mean ± standard deviation (SD). Variable Categories IL-2 (pg/mL) IL-6 (pg/mL) IL-8 (pg/mL) IL-10 (pg/mL) IL-12 (pg/mL) NGF-β (pg/mL) IFN-γ (pg/mL) TNF-α (pg/mL) MCP-1 (pg/mL) VEGF-α (pg/mL) SCF (pg/mL) Sex Male 155.3 ± 330.6 228.0 ± 505.3 1174.7 ± 524.3 38.9 ± 48.3 862.4 ± 1165.3 99.2 ± 169.4 7.1 ± 11.4 52.8 ± 133.6 39.3 ± 21.4 21.8 ± 13.6 159.6 ± 181.4 Female 121.3 ± 266.8 324.2 ± 812.7 1209.9 ± 821.3 58.6 ± 100.8 1311.8 ± 2722.9 72.5 ± 185.8 7.9 ± 11.8 26.5 ± 76.5 62.3 ± 67.2 28.1 ± 23.4 215.3 ± 363.7 q -value (FDR-adjusted) 0.69205 0.59409 0.84889 0.32037 0.38119 0.6112 0.8354 0.47942 0.05029 0.20736 0.4434 Age Puppy 17.1 ± 20.4 37.9 ± 47.9 1025.1 ± 705.1 5.0 ± 37.9 a 542.2 ± 295.1 24.4 ± 57.6 a 6.0 ± 9.0 12.9 ± 28.8 36.6 ± 26.7 a 18.3 ± 17.2 a 79.5 ± 55.2 Young 135.5 ± 177.3 213.4 ± 240.2 1214.2 ± 1226.6 116.3 ± 107.3 b 890.4 ± 595.7 149.0 ± 186.8 b 15.7 ± 18.0 45.0 ± 88.1 38.7 ± 25.2 a 37.4 ± 25.9 b 172.3 ± 135.7 Adult 138.4 ± 325.9 257.9 ± 633.9 1175.3 ± 565.6 26.3 ± 33.8 a 907.9 ± 1256.9 52.4 ± 128.5 a 5.2 ± 8.8 31.2 ± 103.9 50.8 ± 52.2 b 18.4 ± 9.6 a 195.1 ± 349.3 Elder 253.3 ± 449.0 624.2 ± 1269.2 1348.1 ± 597.8 67.87 ± 139.4 b 2463.7 ± 4730.2 102.6 ± 283.6 b 6.2 ± 8.1 38.8 ± 108.9 96.5 ± 90.2 b 37.8 ± 28.2 b 314.6 ± 466.6 q -value (FDR-adjusted) 0.41564 0.29959 0.81737 0.0217 0.195580 < 0.001 0.05972 0.89536 0.041038 0.00613 0.43536 Breed Fonni 196.1 ± 329.4 419.0 ± 897.9 1276.7 ± 917.9 99.1 ± 115.3 1652.1 ± 3331.7 146.2 ± 227.4 11.2 ± 13.7 49.4 ± 94.3 56.6 ± 72.4 37.3 ± 26.2 244.8 ± 334.8 German Shepherd 104.9 ± 298.7 199.1 ± 579.4 1137.1 ± 586.8 15.1 ± 28.4 814.0 ± 1152.9 22.8 ± 102.4 4.8 ± 8.8 19.5 ± 92.4 56.3 ± 47.6 17.4 ± 9.9 164.3 ± 319.9 q -value (FDR-adjusted) 0.27439 0.27967 0.49924 0.00093 0.22215 0.0134 0.04204 0.0233 0.9853 0.00075 0.35177 Due to the absence of seropositive animals, A. phagocytophilum was excluded from further analysis. Seropositivity for E. canis , and Leptospira spp. was not associated with significant changes in cytokine concentrations after FDR correction. Dogs seropositive for L. infantum (Fig. 1 a) and Rickettsia spp. (Fig. 1 b) showed higher serum concentrations of IL-10, NGF-β, and TNF-α compared with seronegative animals ( q < 0.05, FDR-adjusted). Given the low number of L. infantum -seropositive dogs (n = 3), these findings were interpreted with caution. The pairwise interaction analysis revealed a significant interaction between breed and age for IL-10 and IFN-γ concentrations. Young Fonni dogs displayed higher levels of both cytokines compared with age-matched German Shepherds ( q < 0.05, FDR-adjusted) (Fig. 2 ). In addition, an interaction between age and Rickettsia spp. seropositivity was observed for IL-10, NGF-β, and TNF-α, with the highest concentrations detected in seropositive elder dogs ( q < 0.05, FDR-adjusted) (Fig. 3 ). Figure 4 displays pairwise Spearman correlation coefficients (ρ) among serum cytokine values. A significant positive correlation cluster involving IL-6, TNF-α, MCP-1, and VEGF-α (ρ = 0.56–0.78, q < 0.05, FDR-adjusted), indicative of coordinated pro-inflammatory and angiogenic responses. IL-10 showed weak or context-dependent associations with pro-inflammatory cytokines, consistent with a regulatory role. PCA performed on autoscaled cytokine concentrations showed that the first two components explained 57.7% of total variance (PC1 = 32.1% and PC2 = 25.6%). PC1 was mainly driven by proinflammatory and proliferative cytokines (IL-2, IL-6, IL-12, SCF), whereas PC2 was associated with regulatory and modulatory responses (NGF-β, IL-10, TNF-α, IFN-γ). Visualization of PCA scores indicated partial separation according to breed and L. infantum serological status (Fig. 5 a), with Fonni dogs clustering towards higher PC2 values (Fig. 5 b). Discussion This study provides evidence that long-term exposure to endemic vector-borne pathogens can shape breed-specific immune regulation in dogs, with the Fonni breed displaying a profile consistent with infection tolerance rather than inflammatory susceptibility. A limitation of this study is the observational design and the use of privately owned dogs, which may introduce variability in environmental exposure and husbandry conditions. The cytokine panel revealed wide inter-individual variability, yet consistent breed-related patterns emerged. Fonni dogs displayed significantly higher serum levels of IL-10, NGF-β, IFN-γ, TNF-α, and VEGF-α compared to German Shepherds, while MCP-1 was more elevated in adult and elder dogs of both breeds. These results highlight a distinctive immunological profile in the Fonni breed, consistent with previous reports suggesting that Mediterranean native breeds have evolved adaptive mechanisms conferring partial resistance or tolerance to L. infantum infection [ 24 ]. No significant differences were detected between sexes, while age exerted significant effects on IL-10, NGF-β, MCP-1, and VEGF-α concentrations. Young Fonni dogs displayed the highest IL-10 and NGF-β levels, whereas adults and elders showed increased MCP-1 and TNF-α. These findings agreed with studies reporting Th2-polarized responses in immature immune systems [ 25 – 27 ] and inflammation-associated cytokine increases with aging [ 28 , 29 ]. Dogs seropositive for L. infantum and Rickettsia spp. exhibited significantly higher IL-10, NGF-β, and TNF-α levels than uninfected animals. These cytokines are often upregulated during chronic or subclinical infections, where they act to control inflammation [ 30 – 32 ]. The constitutively elevated IL-10 and NGF-β levels observed in Fonni dogs, independent of infection status, suggest a pre-existing regulatory immune phenotype rather than a purely infection-driven response. IL-10 is a potent immunomodulatory cytokine that downregulates Th1-driven inflammation and macrophage activation, thereby reducing tissue damage [ 33 – 35 ]. NGF-β is increasingly recognized as a regulator of macrophage oxidative activity and immune cell survival [ 36 , 37 ]. Experimental models show that NGF-β enhances macrophage hydrogen peroxide production and inhibits L. donovani replication [ 38 ], suggesting a dual role in both parasite control and inflammation limitation. Together with elevated IL-10, this may constitute a tolerance-based immune strategy compatible with a regulatory immune environment that may limit immunopathology under endemic exposure. Such tolerance-based immune strategies have been described in other host-parasite systems and may represent an evolutionarily stable response in an endemic setting, where limiting immunopathology is as critical as controlling parasite replication [ 39 – 41 ]. Fonni dogs also exhibited higher IFN-γ and IL-12 levels than German Shepherds, particularly in young animals. These two cytokines are central to Th1 polarization and macrophage activation [ 42 – 45 ]. Their coexistence with elevated IL-10 indicates a balanced immune regulation capable of controlling intracellular pathogens while minimizing collateral tissue damage. Such an equilibrium has been proposed in other naturally resistant breeds like the Ibizan hound [ 24 ]. IL-6, TNF-α, and MCP-1 formed a correlated pro-inflammatory cluster, as confirmed by Spearman analysis. This cluster likely reflects a coordinated response involving monocyte recruitment and vascular remodelling. VEGF-α, which promotes endothelial permeability and angiogenesis, also correlated with IL-6 and TNF-α, reinforcing its contribution to leukocyte trafficking and tissue repair [ 46 , 47 ]. Fonni dog’s higher VEGF-α levels may therefore support rapid immune cell recruitment without inducing excessive inflammation. The PCA confirmed that cytokine profiles distinguished both infected and uninfected dogs and separated Fonni from German Shepherds along principal components dominated by pro-inflammatory and anti-inflammatory cytokines. This statistical clustering supports the hypothesis that Fonni dogs maintain a distinct immune signature, probably shaped by long-term exposure to Sardinia eco-epidemiological conditions [ 48 – 50 ]. Taken together, the Fonni dog’s cytokine pattern (elevated IL-10, NGF-β, IFN-γ, TNF-α, and VEGF-α) supports the existence of an evolved immune tolerance mechanism that is compatible with a regulatory immune environment that may limit immunopathology in endemic settings. Similar adaptive strategies have been proposed by Llobat and colleagues in other Mediterranean breeds exposed to endemic pathogens, highlighting the influence of genetic background and environmental selection on canine immune profiles [ 17 , 24 , 51 ]. The combination of regulatory (IL-10, NGF-β) and protective (IL-12, IFN-γ) cytokines indicates a finely tuned immune equilibrium that could be harnessed for vaccine development or immunomodulatory therapies aimed at achieving consistent results with reduced immunopathology under endemic exposure. Conclusions In an endemic Mediterranean setting, the Fonni dog exhibits a distinct immune signature indicative of tolerance-like immune phenotype under endemic exposure to Leishmania infantum , rather than susceptibility driven by excessive inflammation. The correlation between pro-inflammatory (IL-6, TNF-α, and MCP-1) and angiogenic (VEGF-α) mediators further suggests coordinated regulation of inflammation and tissue repair. The Fonni dog exhibits a distinct cytokine and growth factor signature characterized by elevated IL-10, NGF-β, IFN-γ, TNF-α, and VEGF-α concentrations, supporting a tolerant immune strategy against L. infantum infection. Fonni dogs appear capable of modulating their immune response in a manner compatible with limited immunopathology under endemic exposure. Overall, these findings reinforce previous evidence that breed-specific immune adaptations strongly influence the outcome of L. infantum infection in dogs. Understanding these natural resistance mechanisms in native breeds such as the Fonni dogs may offer valuable models for developing sustainable control strategies for zoonotic leishmaniosis. Declarations CRediT authorship contribution statement Lola Martínez-Sáez: Data curation, methodology, formal analysis, writing – original draft; Raffaella Cocco: Data curation; Luigi Liotta: Data curation, writing-review and editing. Pablo Jesús Marín-García: methodology, formal analysis; Lola Llobat: Writing – review & editing, Writing – original draft, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis, Conceptualization. Funding information This research was funded by Universidad Cardenal Herrera, grant numbers INDI25-42, and GIR25-32. Lola Martínez-Sáez is supported by a Predoctoral Contract from the Universidad Cardenal Herrera CEU. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. Declaration of Competing Interest The authors declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article. Ethical considerations All procedures complied with European Directive 2010/63/EU on animal protection. Acknowledgements We are grateful to the Universidad Cardenal Herrera CEU (Spain), Universita ` degli Studi di Messina (Italy) and University of Sassari (Italy). This paper is part of the Doctoral Thesis of Lola Martínez-Sáez, and all authors agree. References Costa CHN, Chang K-P, Costa DL, Cunha FVM. From Infection to Death: An Overview of the Pathogenesis of Visceral Leishmaniasis. Pathogens. 2023;12:969. https://doi.org/10.3390/pathogens12070969 Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis Worldwide and Global Estimates of Its Incidence. PLOS ONE. 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Su X, Yu Y, Zhong Y, Giannopoulou EG, Hu X, Liu H, et al. Interferon-γ regulates cellular metabolism and mRNA translation to potentiate macrophage activation. Nat Immunol. 2015;16:838–49. https://doi.org/10.1038/ni.3205 Ullrich KA-M, Schulze LL, Paap E-M, Müller TM, Neurath MF, Zundler S. Immunology of IL-12: An update on functional activities and implications for disease. EXCLI J. 2020;19:1563–89. https://doi.org/10.17179/excli2020-3104 Sato T, Takeuchi M, Karasawa Y, Takayama K, Enoki T. Comprehensive expression patterns of inflammatory cytokines in aqueous humor of patients with neovascular age-related macular degeneration. Sci Rep. 2019;9:19447. https://doi.org/10.1038/s41598-019-55191-x Obadă O, Pantalon AD, Rusu-Zota G, Hăisan A, Lupuşoru SI, Constantinescu D, et al. Aqueous Humor Cytokines in Non-Proliferative Diabetic Retinopathy. Medicina (Kaunas). 2022;58:909. https://doi.org/10.3390/medicina58070909 Tamponi C, Scarpa F, Carta S, Knoll S, Sanna D, Gai C, et al. Seroprevalence and risk factors associated with Leishmania infantum in dogs in Sardinia (Italy), an endemic island for leishmaniasis. Parasitol Res. 2021;120:289–300. https://doi.org/10.1007/s00436-020-06973-0 Cortellari M, Bionda A, Cocco R, Sechi S, Liotta L, Crepaldi P. Genomic Analysis of the Endangered Fonni’s Dog Breed: A Comparison of Genomic and Phenotypic Evaluation Scores. Animals (Basel). 2023;13:818. https://doi.org/10.3390/ani13050818 Cocco R, Sechi S, Rizzo M, Bonomo A, Arfuso F, Giudice E. Haematochemical Profile of Healthy Dogs Seropositive for Single or Multiple Vector-Borne Pathogens. Vet Sci. 2024;11:205. https://doi.org/10.3390/vetsci11050205 Álvarez L, Marín-García P-J, Llobat L. Genetic haplotypes associated with immune response to Leishmania infantum infection in dogs. Vet Res Commun. 2023;47:1675–85. https://doi.org/10.1007/s11259-023-10123-z Additional Declarations No competing interests reported. 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16:07:22","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161223,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8367154/v1/c45c0e07998ddb4ca12bc7a6.html"},{"id":98821340,"identity":"3d50872f-5849-40a4-acff-aa5112977504","added_by":"auto","created_at":"2025-12-22 17:21:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50221,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine concentrations in \u003cem\u003eL. infantum \u003c/em\u003e(a)\u003cem\u003e and Rickettsia \u003c/em\u003espp.\u003cem\u003e \u003c/em\u003e(b) positive and negative dogs. Boxplots show the values of cytokines statistically different between the two groups (IL-10, NGF-β and TNF-α). \u003cem\u003eL. infantum\u003c/em\u003e and \u003cem\u003eRickettsia \u003c/em\u003espp.\u003cem\u003e \u003c/em\u003epositive animals presented significantly higher IL-10, NGF-β and TNF-α values compared with uninfected animals (\u003cem\u003eq \u003c/em\u003e\u0026lt; 0.05, Benjamini-Hochberg FDR-adjusted). Results involving \u003cem\u003eL. infantum\u003c/em\u003eseropositive dogs should be interpreted with caution due to the low number of positive animals (n=3). IL: interleukin, NGF: nerve growth factor, TNF: tumor necrosis factor.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8367154/v1/4296f13149939a930b6ed510.png"},{"id":98821338,"identity":"c1f0983f-e082-4a90-b82d-c2fd5ca8010e","added_by":"auto","created_at":"2025-12-22 17:21:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42815,"visible":true,"origin":"","legend":"\u003cp\u003eIL-10 (a) and IFN-γ (b) serum levels according to age-group and breed. Boxplots show the values of cytokines statistically different between the groups. Young animals of the Fonni breed presented the highest values of these two cytokines (\u003cem\u003eq \u003c/em\u003e\u0026lt; 0.05, Benjamini-Hochberg FDR-adjusted). Results involving \u003cem\u003eL. infantum\u003c/em\u003eseropositive dogs should be interpreted with caution due to the low number of positive animals (n=3). IL: interleukin, IFN: interferon.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8367154/v1/9c9da67b234acc2b02f04d7e.png"},{"id":99307999,"identity":"463300fe-0670-411e-9b00-9311b440dcd2","added_by":"auto","created_at":"2025-12-31 16:07:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48536,"visible":true,"origin":"","legend":"\u003cp\u003eIL-10 (a), NGF-β (b), and TNF-α (c) serum levels according to age-group in infected (positive) and uninfected (negative) dogs. Boxplots show the values of cytokines statistically different between the groups. Elder infected dogs presented the highest values of these three cytokines (\u003cem\u003eq \u003c/em\u003e\u0026lt; 0.05, Benjamini-Hochberg FDR-adjusted). Results involving \u003cem\u003eL. infantum\u003c/em\u003e seropositive dogs should be interpreted with caution due to the low number of positive animals (n=3). IL: interleukin, NGF: nerve growth factor, TNF: tumor necrosis factor.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8367154/v1/4444bc5c2e7b7c15afae5565.png"},{"id":98821348,"identity":"fcc57d37-309a-4ce2-9b64-d9ab666b8550","added_by":"auto","created_at":"2025-12-22 17:21:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":456709,"visible":true,"origin":"","legend":"\u003cp\u003eSpearman correlation matrix among cytokines. Color intensity indicates the strength and direction of Spearman correlations (ρ) among cytokines. Asterisks denote statistically significant correlations after Benjamini-Hochberg false discovery rate (FDR) correction (\u003cem\u003eq\u003c/em\u003e-value \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8367154/v1/2139198f7b8d136062a9c82d.png"},{"id":98821346,"identity":"420a83db-54d5-4c9a-a051-52bef30cda73","added_by":"auto","created_at":"2025-12-22 17:21:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":950439,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis PCA of cytokine and immune factor profiles. PCA was conducted using standardized concentrations of eleven cytokines (IL-2, IL-6, IL-8, IL-10, IL-12, NGF-β, IFN-γ, TNF-α, MCP-1, VEGF-α, and SCF) only. Categorical variables (breed and infection status) were used exclusively for visualization and were not included in the PCA computation. Each point represents an individual dog, while shaded areas indicate the distribution of samples within each group. a) Cluster according to \u003cem\u003eL. infantum\u003c/em\u003e infection status, and b) cluster according to dog breed. Given the low number of \u003cem\u003eL. infantum\u003c/em\u003e-seropositive dogs (n=3), clustering by infection status should be interpreted cautiously. IL: interleukin, NGF: nerve growth factor, IFN: interferon, TNF: tumor necrosis factor, MCP: monocyte chemoattractant protein, VEGF: vascular endothelial growth factor, SCF: stem cell factor.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8367154/v1/17223644bca8ddb7367421a6.png"},{"id":104739628,"identity":"1b863dcb-e0d2-4596-9a1a-e4a43939b59e","added_by":"auto","created_at":"2026-03-16 16:10:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1976420,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8367154/v1/46451907-b3df-41a1-a73b-50d90d6f7f28.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Breed-specific immune tolerance to Leishmania infantum in a native Mediterranean canine population","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVector-borne diseases (VBDs) are among the most significant infectious threats to domestic dogs worldwide, particularly in the Mediterranean basin, where climatic conditions favor the proliferation of phlebotomine sandflies, ticks, and fleas that transmit protozoal, bacterial, and rickettsial agents. Among these pathogens, \u003cem\u003eLeishmania infantum\u003c/em\u003e is the etiological agent of zoonotic visceral leishmaniasis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], a disease endemic to Africa, Asia, the Americas, and Europe [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Within Europe, leishmaniasis persists in the Mediterranean region, particularly in Spain and Italy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], where the domestic dog (\u003cem\u003eCanis lupus familiaris\u003c/em\u003e) serves as the primary reservoir host [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, dogs in these regions are often exposed not only to \u003cem\u003eL. infantum\u003c/em\u003e, but also to other vector-borne pathogens such as \u003cem\u003eAnaplasma phagocytophilum\u003c/em\u003e, \u003cem\u003eEhrlichia canis\u003c/em\u003e, \u003cem\u003eLeptospira\u003c/em\u003e spp., and \u003cem\u003eRickettsia\u003c/em\u003e spp., which may circulate simultaneously in overlapping ecological niches [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Co-infections or sequential exposures to multiple VBDs can modulate host immunity, potentially affecting both susceptibility to \u003cem\u003eL. infantum\u003c/em\u003e and the clinical outcome of infection [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The immunopathogenesis of canine leishmaniosis depends largely on the host\u0026rsquo;s ability to mount a protective Th1 immune response, characterized by IFN-γ, IL-2, and IL-12 production, while disease progression is often associated with elevated IL-10 and other cytokines related to anti-inflammatory and angiogenic activity [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, some Mediterranean dog breeds show a natural resistance to \u003cem\u003eL. infantum\u003c/em\u003e infection. For instance, the Ibizan hound and Cirneco dell\u0026rsquo;Etna display enhanced pro-inflammatory cytokine profiles and genetic polymorphisms in immune-related genes such as \u003cem\u003eIFNG\u003c/em\u003e and \u003cem\u003eIL6R\u003c/em\u003e, which appear to confer partial protection against infection [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This genetic and immunological diversity suggests that host factors play a critical role in disease progression. Nonetheless, little is known about other native Mediterranean breeds, including the Fonni\u0026rsquo;s dogs (\u003cem\u003eCane Fonnese\u003c/em\u003e), a rustic Sardinian molossoid breed recognized by the Italian Kennel Club (ENCI). Archaeological evidence suggests its origin dates to the Bronze Age, highlighting its long-standing adaptation to the Mediterranean pathogens [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSardinia represents a unique epidemiological setting, where \u003cem\u003eL. infantum\u003c/em\u003e coexists with a high prevalence of other vector-borne pathogens [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In such ecosystems, assessing the immune responses of local breeds may offer valuable insights into the mechanisms underlying natural resistance to infection.\u003c/p\u003e \u003cp\u003eIn this context, we investigated whether a native Mediterranean canine population chronically exposed to multiple vector-borne pathogens displays an immune profile consistent with tolerance to \u003cem\u003eLeishmania infantum\u003c/em\u003e, using the Fonni dog as a naturally adapted host and the German Shepherds as a comparative breed.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e This study is reported in accordance with the ARRIVE guidelines for animal research, including detailed descriptions of animal characteristics, inclusion and exclusion criteria, housing conditions, and statistical methods.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population and sampling\u003c/h2\u003e \u003cp\u003eA cross-sectional observational study was conducted between September 2024 and April 2025 in Sardinia (Italy), a Mediterranean island endemic for \u003cem\u003eL. infantum\u003c/em\u003e and other canine vector-borne pathogens. Fifty-nine privately owned, clinically healthy domestic dogs were included. Dogs were recruited consecutively during routine veterinary clinical visits, and the sample size corresponded to all animals meeting the inclusion criteria during the study period; therefore, no formal \u003cem\u003ea priori\u003c/em\u003e sample size calculation was performed.\u003c/p\u003e \u003cp\u003eInclusion criteria comprised the absence of clinical signs compatible with canine leishmaniosis or other systemic infectious disease at the time of sampling, and owner consent to participate. Dogs showing overt clinical illness were excluded. All owners provided written informed consent before inclusion.\u003c/p\u003e \u003cp\u003eEpidemiological data recorded for each animal included sex (male/female), age, and breed. Dogs were grouped by age into four categories: puppies (2\u0026ndash;12 months), young (1\u0026ndash;2 years), adults (3\u0026ndash;8 years), and elder (9\u0026ndash;11 years). Only dogs with documented pedigree certification were included in the breed analysis, which comprised the Fonni dog (\u003cem\u003eCane Fonnese\u003c/em\u003e) and German Shepherds.\u003c/p\u003e \u003cp\u003eDogs were maintained under typical household conditions according to owner routines, with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. Information on antiparasitic prophylaxis was obtained from owners and clinical records. None of the enrolled dogs had received topical pyrethroid-based repellents or insecticidal collars in the 12 months preceding sampling. No animal had received immunosuppressive therapy during this period. The absence of standardized vector control measures was considered representative of natural exposure conditions in the study area.\u003c/p\u003e \u003cp\u003eAll samples were obtained as part of routine clinical practice, and no procedures were performed specifically for research purposes. Blood collection was carried out by licensed veterinarians, required less than three minutes per animal, and involved gentle manual restraint to minimize stress. All samples and associated data were anonymized before analysis. According to institutional and national regulations, and in line with European Directive 2010/63/EU on the protection of animals used for scientific purposes, formal ethical committee approval was not required for the use of surplus clinical samples not affecting patient management.\u003c/p\u003e \u003cp\u003eFor each dog, ten milliliters of blood were obtained by jugular venipuncture and divided into two tubes, one with EDTA to perform DNA extraction, and another without anticoagulant for serum separation. The tubes were maintained at room temperature. Serum samples were obtained by centrifuging the non-anticoagulated blood at 3000 rpm for 10 minutes. The separated serum was transferred to cryotubes and stored at -80\u0026ordm;C until infection and cytokine determination.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInfection determination\u003c/h3\u003e\n\u003cp\u003eSerum samples were analyzed to detect antibodies against \u003cem\u003eA. phagocytophilum\u003c/em\u003e, \u003cem\u003eE. canis\u003c/em\u003e, \u003cem\u003eL. infantum\u003c/em\u003e, \u003cem\u003eLeptospira\u003c/em\u003e spp., and \u003cem\u003eRickettsia\u003c/em\u003e spp. Antibody detection was performed using the indirect immunofluorescence assay (IFAT) following the specific protocols for each pathogen.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eA. phagocytophilum\u003c/em\u003e, slides containing the corresponding antigen (Fuller Laboratories) were used, and titers greater than 1:40 were considered positive. Detection of anti-\u003cem\u003eE. canis\u003c/em\u003e antibodies followed the protocol described by [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe presence of antibodies against \u003cem\u003eLeptospira\u003c/em\u003e spp. was determined using the Microscopic Agglutination Test (MAT), which is considered the reference serological method for diagnosing leptospirosis. The test was performed according to the standard procedures recommended by the World Organization for Animal Health (OIE). A panel of live \u003cem\u003eLeptospira\u003c/em\u003e reference serovars representing the main pathogenic serogroups circulating in the region was used as antigens. Serum samples were initially diluted 1:50 in phosphate-buffered saline (PBS) and subsequently mixed with an equal volume of each live antigen suspension. After incubation at 28\u0026ndash;30\u0026ordm;C for 1 hour, the mixtures were examined under a dark-field microscope to assess agglutination. A sample was considered positive when \u0026ge;\u0026thinsp;50% of the leptospires were agglutinated compared to the control suspension. Positive sera were further titrated through serial two-fold dilutions to determine the highest dilution showing\u0026thinsp;\u0026ge;\u0026thinsp;50% agglutination, which was recorded as the antibody titer. Titers\u0026thinsp;\u0026ge;\u0026thinsp;1:100 were considered positive.\u003c/p\u003e \u003cp\u003eFinally, anti-\u003cem\u003eL. infantum\u003c/em\u003e IgG antibodies were detected using an in-house IFA in accordance with the World Organisation for Animal Health (WOAH, formerly OIE). Titers\u0026thinsp;\u0026ge;\u0026thinsp;1:80 were considered positive for both pathogens, and anti-\u003cem\u003eRickettsia\u003c/em\u003e spp. IgG antibodies were detected using a commercial canine \u003cem\u003eRickettsia\u003c/em\u003e spp. IgG Indirect Fluorescent Antibody Test (IFAT) kit, applying a positivity threshold of \u0026ge;\u0026thinsp;1:128, following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eCytokine quantification\u003c/h3\u003e\n\u003cp\u003eSerum levels of IL-2, IL-6, IL-8 (CXCL8), IL-10, IL-12/IL-23p40, NGF-β, IFN-γ, TNF-α, MCP-1 (CCL2), VEGF-α and SCF were quantified using the ProcartaPlex\u0026trade; Canine Cytokine/Chemokine/Growth Factor Panel (ThermoFisher, Scientific, Waltham, MA, USA). Analyses were performed using the Luminex\u0026trade; 200X detection system in serum samples, following the manufacturer's instructions. All samples were analyzed in duplicate, and intra-assay and inter-assay coefficients of variation were \u0026lt;\u0026thinsp;10% and \u0026lt;\u0026thinsp;15%, respectively. Cytokine values below the detection limit were replaced with half of the minimum detectable concentration.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SAS software (version 9.2, North Carolina State University, USA). Cytokine concentrations were log10-transformed before analysis to reduce skewness. Linear mixed-effects models (PROC MIXED, REML estimation) were applied to evaluate the effects of breed, age group, sex, and serological status for each pathogen as fixed effects. Pairwise interactions between breed and age, and between breed and infection status, were included when biologically relevant. No random effects were specified, as each dog contributed a single observation. Model assumptions were assessed through inspection of residuals. \u003cem\u003eP\u003c/em\u003e-values were adjusted for multiple testing across cytokines using the Benjamini-Hochberg false discovery rate (FDR), with adjusted \u003cem\u003eq\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e \u003cp\u003eDescriptive statistics (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, median, range) were calculated for all cytokines. Correlations among cytokine concentrations were evaluated using Spearman\u0026rsquo;s rank correlation coefficient (ρ), and correlation matrices were visualized as color-coded heatmaps with labelled \u003cem\u003ep\u003c/em\u003e-values.\u003c/p\u003e \u003cp\u003ePrincipal component analysis (PCA) was performed using MetaboAnalyst on autoscaled cytokine concentrations only. Categorical variables (breed, age group, infection status) were used exclusively for sample annotation and visualization and were not included in the PCA computation. to assess the quality of the dataset, identify potential outliers, and evaluate differences among the experimental groups. Before any analysis, all datasets were preprocessed using autoscaling, followed by normalization to the median [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eOf the 59 dogs included in the study, fifteen were males (25.4%) and forty-four were females (74.6%). According to age, 8 dogs were classified as puppies (13.6%), 12 as young (20.3%), 26 as adults (44.1%), and 13 as elders (22.0%). Twenty-seven dogs belonged to the Fonni breed (45.8%), and thirty-two were German Shepherd (54.2%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Serological screening revealed antibodies against \u003cem\u003eRickettsia\u003c/em\u003e spp. in 21 dogs (35.6%), \u003cem\u003eLeptospira\u003c/em\u003e spp. in 6 dogs (10.2%), \u003cem\u003eE. canis\u003c/em\u003e in 3 dogs (5.1%), and \u003cem\u003eL. infantum\u003c/em\u003e in 3 dogs (5.1%). No dogs were seropositive for \u003cem\u003eA. phagocytophilum\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Serum cytokine and growth factor concentrations exhibited wide inter-individual variability. Median values were lowest for IFN-γ and highest for IL-8 and IL-12 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After log10 transformation, residual inspection indicated acceptable model fit for all analytes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEpidemiological data of dogs are included in the study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCategories\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of dogs (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (25.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44 (74.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePuppy (2\u0026ndash;12 months)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (13.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYoung (1\u0026ndash;2 years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12 (20.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdults (3\u0026ndash;8 years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 (44.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElder (9\u0026ndash;11 years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (22.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBreed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFonni\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27 (45.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGerman Shepherd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32 (54.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eA. \u003cem\u003ephagocytophilum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59 (100%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. canis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56 (94.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLeptospira\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (10.2%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53 (89.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eL. infantum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56 (94.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRickettsia\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21 (35.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38 (64.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean and standard deviation of all cytokines analyzed. IL: interleukin; NGF: nerve growth factor; IFN: interferon; TNF: tumor necrosis factor; MCP: monocyte chemoattractant protein; VEGF: vascular endothelial growth factor; SCF: stem cell factor. Cytokine serum levels are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIL-2\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIL-6\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIL-8\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIL-10\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIL-12\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNGF-β (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIFN-γ (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTNF-α (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMCP-1\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eVEGF-α (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eSCF\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e146.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e299.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1200.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1197.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e79.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e7.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e33.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e56.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e26.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e201.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e313.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e743.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e752.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e90.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2421.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e180.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e11.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e93.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e59.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e21.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e326.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eRegarding fixed effects, no statistical differences were found between males and females; however, age exerted a significant effect on selected mediators (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). IL-10 and NGF-β concentrations were highest in young dogs, whereas MCP-1 increased progressively with age, reaching the highest values in adult and elder animals (\u003cem\u003eq\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FDR-adjusted). VEGF-α also varied significantly across age groups, with higher concentrations in young and elder dogs compared with puppies. Marked breed-associated differences were observed (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Fonni dogs exhibited significantly higher serum concentrations of IL-10 (99.1\u0026thinsp;\u0026plusmn;\u0026thinsp;115.3 pg/mL), NGF-β (146.2\u0026thinsp;\u0026plusmn;\u0026thinsp;227.4 pg/mL), IFN-γ (11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;13.7 pg/mL), TNF-α (49.4\u0026thinsp;\u0026plusmn;\u0026thinsp;94.3 pg/mL) and VEGF-α (37.3\u0026thinsp;\u0026plusmn;\u0026thinsp;26.2 pg/mL) than German Shepherd dogs (15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;28.4, 22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;102.4, 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8, 19.5\u0026thinsp;\u0026plusmn;\u0026thinsp;92.4 pg/mL and 17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.9 pg/mL, respectively). No breed-related differences were detected for IL-2, IL-6, IL-8, IL-12, MCP-1, or SCF.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCytokine serum levels according to the variables. Bold numbers show statistical differences (\u003cem\u003eq\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Benjamini-Hochberg FDR-adjusted) in cytokine serum levels. Different superscripts in the same row indicate statistical differences. \u003cem\u003eP\u003c/em\u003e-values correspond to linear mixed-effects models on log-transformed data with Benjamini-Hochberg FDR correction. IL: interleukin; NGF: nerve growth factor; IFN: interferon; TNF: tumor necrosis factor; MCP: monocyte chemoattractant protein; VEGF: vascular endothelial growth factor; SCF: stem cell factor. Cytokine serum levels are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCategories\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIL-2\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIL-6\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIL-8\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIL-10\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIL-12\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNGF-β (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eIFN-γ (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eTNF-α (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eMCP-1\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eVEGF-α (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eSCF\u003c/p\u003e \u003cp\u003e(pg/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e155.3\u0026thinsp;\u0026plusmn;\u0026thinsp;330.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e228.0\u0026thinsp;\u0026plusmn;\u0026thinsp;505.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1174.7\u0026thinsp;\u0026plusmn;\u0026thinsp;524.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.9\u0026thinsp;\u0026plusmn;\u0026thinsp;48.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e862.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1165.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99.2\u0026thinsp;\u0026plusmn;\u0026thinsp;169.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;11.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e52.8\u0026thinsp;\u0026plusmn;\u0026thinsp;133.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e39.3\u0026thinsp;\u0026plusmn;\u0026thinsp;21.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;13.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e159.6\u0026thinsp;\u0026plusmn;\u0026thinsp;181.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e121.3\u0026thinsp;\u0026plusmn;\u0026thinsp;266.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e324.2\u0026thinsp;\u0026plusmn;\u0026thinsp;812.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1209.9\u0026thinsp;\u0026plusmn;\u0026thinsp;821.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e58.6\u0026thinsp;\u0026plusmn;\u0026thinsp;100.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1311.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2722.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e72.5\u0026thinsp;\u0026plusmn;\u0026thinsp;185.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;11.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e26.5\u0026thinsp;\u0026plusmn;\u0026thinsp;76.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e62.3\u0026thinsp;\u0026plusmn;\u0026thinsp;67.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;23.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e215.3\u0026thinsp;\u0026plusmn;\u0026thinsp;363.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eq\u003c/em\u003e-value (FDR-adjusted)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.69205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.59409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.84889\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.32037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.38119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.6112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.8354\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.47942\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.05029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.20736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.4434\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePuppy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.1\u0026thinsp;\u0026plusmn;\u0026thinsp;20.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.9\u0026thinsp;\u0026plusmn;\u0026thinsp;47.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1025.1\u0026thinsp;\u0026plusmn;\u0026thinsp;705.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;37.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e542.2\u0026thinsp;\u0026plusmn;\u0026thinsp;295.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24.4\u0026thinsp;\u0026plusmn;\u0026thinsp;57.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.9\u0026thinsp;\u0026plusmn;\u0026thinsp;28.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e36.6\u0026thinsp;\u0026plusmn;\u0026thinsp;26.7\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;17.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e79.5\u0026thinsp;\u0026plusmn;\u0026thinsp;55.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYoung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e135.5\u0026thinsp;\u0026plusmn;\u0026thinsp;177.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e213.4\u0026thinsp;\u0026plusmn;\u0026thinsp;240.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1214.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1226.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e116.3\u0026thinsp;\u0026plusmn;\u0026thinsp;107.3\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e890.4\u0026thinsp;\u0026plusmn;\u0026thinsp;595.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e149.0\u0026thinsp;\u0026plusmn;\u0026thinsp;186.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e15.7\u0026thinsp;\u0026plusmn;\u0026thinsp;18.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e45.0\u0026thinsp;\u0026plusmn;\u0026thinsp;88.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e38.7\u0026thinsp;\u0026plusmn;\u0026thinsp;25.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e37.4\u0026thinsp;\u0026plusmn;\u0026thinsp;25.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e172.3\u0026thinsp;\u0026plusmn;\u0026thinsp;135.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdult\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e138.4\u0026thinsp;\u0026plusmn;\u0026thinsp;325.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e257.9\u0026thinsp;\u0026plusmn;\u0026thinsp;633.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1175.3\u0026thinsp;\u0026plusmn;\u0026thinsp;565.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26.3\u0026thinsp;\u0026plusmn;\u0026thinsp;33.8\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e907.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1256.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e52.4\u0026thinsp;\u0026plusmn;\u0026thinsp;128.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e31.2\u0026thinsp;\u0026plusmn;\u0026thinsp;103.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e50.8\u0026thinsp;\u0026plusmn;\u0026thinsp;52.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e18.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e195.1\u0026thinsp;\u0026plusmn;\u0026thinsp;349.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e253.3\u0026thinsp;\u0026plusmn;\u0026thinsp;449.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e624.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1269.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1348.1\u0026thinsp;\u0026plusmn;\u0026thinsp;597.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e67.87\u0026thinsp;\u0026plusmn;\u0026thinsp;139.4\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2463.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4730.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e102.6\u0026thinsp;\u0026plusmn;\u0026thinsp;283.6\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e38.8\u0026thinsp;\u0026plusmn;\u0026thinsp;108.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e96.5\u0026thinsp;\u0026plusmn;\u0026thinsp;90.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e37.8\u0026thinsp;\u0026plusmn;\u0026thinsp;28.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e314.6\u0026thinsp;\u0026plusmn;\u0026thinsp;466.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eq\u003c/em\u003e-value (FDR-adjusted)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.41564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.29959\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.81737\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.0217\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.195580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.05972\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.89536\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e0.041038\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e0.00613\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.43536\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBreed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFonni\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e196.1\u0026thinsp;\u0026plusmn;\u0026thinsp;329.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e419.0\u0026thinsp;\u0026plusmn;\u0026thinsp;897.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1276.7\u0026thinsp;\u0026plusmn;\u0026thinsp;917.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.1\u0026thinsp;\u0026plusmn;\u0026thinsp;115.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1652.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3331.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e146.2\u0026thinsp;\u0026plusmn;\u0026thinsp;227.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;13.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e49.4\u0026thinsp;\u0026plusmn;\u0026thinsp;94.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e56.6\u0026thinsp;\u0026plusmn;\u0026thinsp;72.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e37.3\u0026thinsp;\u0026plusmn;\u0026thinsp;26.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e244.8\u0026thinsp;\u0026plusmn;\u0026thinsp;334.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGerman Shepherd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e104.9\u0026thinsp;\u0026plusmn;\u0026thinsp;298.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e199.1\u0026thinsp;\u0026plusmn;\u0026thinsp;579.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1137.1\u0026thinsp;\u0026plusmn;\u0026thinsp;586.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;28.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e814.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1152.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;102.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e19.5\u0026thinsp;\u0026plusmn;\u0026thinsp;92.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e56.3\u0026thinsp;\u0026plusmn;\u0026thinsp;47.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e164.3\u0026thinsp;\u0026plusmn;\u0026thinsp;319.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eq\u003c/em\u003e-value (FDR-adjusted)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.27439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.27967\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.49924\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e0.00093\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.22215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.0134\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e0.04204\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e0.0233\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.9853\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cb\u003e0.00075\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.35177\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDue to the absence of seropositive animals, \u003cem\u003eA. phagocytophilum\u003c/em\u003e was excluded from further analysis. Seropositivity for \u003cem\u003eE. canis\u003c/em\u003e, and \u003cem\u003eLeptospira\u003c/em\u003e spp. was not associated with significant changes in cytokine concentrations after FDR correction. Dogs seropositive for \u003cem\u003eL. infantum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) and \u003cem\u003eRickettsia\u003c/em\u003e spp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) showed higher serum concentrations of IL-10, NGF-β, and TNF-α compared with seronegative animals (\u003cem\u003eq\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FDR-adjusted). Given the low number of \u003cem\u003eL. infantum\u003c/em\u003e-seropositive dogs (n\u0026thinsp;=\u0026thinsp;3), these findings were interpreted with caution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe pairwise interaction analysis revealed a significant interaction between breed and age for IL-10 and IFN-γ concentrations. Young Fonni dogs displayed higher levels of both cytokines compared with age-matched German Shepherds (\u003cem\u003eq\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FDR-adjusted) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, an interaction between age and \u003cem\u003eRickettsia\u003c/em\u003e spp. seropositivity was observed for IL-10, NGF-β, and TNF-α, with the highest concentrations detected in seropositive elder dogs (\u003cem\u003eq\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FDR-adjusted) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e displays pairwise Spearman correlation coefficients (ρ) among serum cytokine values. A significant positive correlation cluster involving IL-6, TNF-α, MCP-1, and VEGF-α (ρ\u0026thinsp;=\u0026thinsp;0.56\u0026ndash;0.78, \u003cem\u003eq\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FDR-adjusted), indicative of coordinated pro-inflammatory and angiogenic responses. IL-10 showed weak or context-dependent associations with pro-inflammatory cytokines, consistent with a regulatory role.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePCA performed on autoscaled cytokine concentrations showed that the first two components explained 57.7% of total variance (PC1\u0026thinsp;=\u0026thinsp;32.1% and PC2\u0026thinsp;=\u0026thinsp;25.6%). PC1 was mainly driven by proinflammatory and proliferative cytokines (IL-2, IL-6, IL-12, SCF), whereas PC2 was associated with regulatory and modulatory responses (NGF-β, IL-10, TNF-α, IFN-γ). Visualization of PCA scores indicated partial separation according to breed and \u003cem\u003eL. infantum\u003c/em\u003e serological status (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), with Fonni dogs clustering towards higher PC2 values (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides evidence that long-term exposure to endemic vector-borne pathogens can shape breed-specific immune regulation in dogs, with the Fonni breed displaying a profile consistent with infection tolerance rather than inflammatory susceptibility. A limitation of this study is the observational design and the use of privately owned dogs, which may introduce variability in environmental exposure and husbandry conditions. The cytokine panel revealed wide inter-individual variability, yet consistent breed-related patterns emerged. Fonni dogs displayed significantly higher serum levels of IL-10, NGF-β, IFN-γ, TNF-α, and VEGF-α compared to German Shepherds, while MCP-1 was more elevated in adult and elder dogs of both breeds. These results highlight a distinctive immunological profile in the Fonni breed, consistent with previous reports suggesting that Mediterranean native breeds have evolved adaptive mechanisms conferring partial resistance or tolerance to \u003cem\u003eL. infantum\u003c/em\u003e infection [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNo significant differences were detected between sexes, while age exerted significant effects on IL-10, NGF-β, MCP-1, and VEGF-α concentrations. Young Fonni dogs displayed the highest IL-10 and NGF-β levels, whereas adults and elders showed increased MCP-1 and TNF-α. These findings agreed with studies reporting Th2-polarized responses in immature immune systems [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and inflammation-associated cytokine increases with aging [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Dogs seropositive for \u003cem\u003eL. infantum\u003c/em\u003e and \u003cem\u003eRickettsia\u003c/em\u003e spp. exhibited significantly higher IL-10, NGF-β, and TNF-α levels than uninfected animals. These cytokines are often upregulated during chronic or subclinical infections, where they act to control inflammation [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe constitutively elevated IL-10 and NGF-β levels observed in Fonni dogs, independent of infection status, suggest a pre-existing regulatory immune phenotype rather than a purely infection-driven response. IL-10 is a potent immunomodulatory cytokine that downregulates Th1-driven inflammation and macrophage activation, thereby reducing tissue damage [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. NGF-β is increasingly recognized as a regulator of macrophage oxidative activity and immune cell survival [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Experimental models show that NGF-β enhances macrophage hydrogen peroxide production and inhibits \u003cem\u003eL. donovani\u003c/em\u003e replication [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], suggesting a dual role in both parasite control and inflammation limitation. Together with elevated IL-10, this may constitute a tolerance-based immune strategy compatible with a regulatory immune environment that may limit immunopathology under endemic exposure.\u003c/p\u003e \u003cp\u003eSuch tolerance-based immune strategies have been described in other host-parasite systems and may represent an evolutionarily stable response in an endemic setting, where limiting immunopathology is as critical as controlling parasite replication [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Fonni dogs also exhibited higher IFN-γ and IL-12 levels than German Shepherds, particularly in young animals. These two cytokines are central to Th1 polarization and macrophage activation [\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Their coexistence with elevated IL-10 indicates a balanced immune regulation capable of controlling intracellular pathogens while minimizing collateral tissue damage. Such an equilibrium has been proposed in other naturally resistant breeds like the Ibizan hound [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIL-6, TNF-α, and MCP-1 formed a correlated pro-inflammatory cluster, as confirmed by Spearman analysis. This cluster likely reflects a coordinated response involving monocyte recruitment and vascular remodelling. VEGF-α, which promotes endothelial permeability and angiogenesis, also correlated with IL-6 and TNF-α, reinforcing its contribution to leukocyte trafficking and tissue repair [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Fonni dog\u0026rsquo;s higher VEGF-α levels may therefore support rapid immune cell recruitment without inducing excessive inflammation.\u003c/p\u003e \u003cp\u003eThe PCA confirmed that cytokine profiles distinguished both infected and uninfected dogs and separated Fonni from German Shepherds along principal components dominated by pro-inflammatory and anti-inflammatory cytokines. This statistical clustering supports the hypothesis that Fonni dogs maintain a distinct immune signature, probably shaped by long-term exposure to Sardinia eco-epidemiological conditions [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTaken together, the Fonni dog\u0026rsquo;s cytokine pattern (elevated IL-10, NGF-β, IFN-γ, TNF-α, and VEGF-α) supports the existence of an evolved immune tolerance mechanism that is compatible with a regulatory immune environment that may limit immunopathology in endemic settings. Similar adaptive strategies have been proposed by Llobat and colleagues in other Mediterranean breeds exposed to endemic pathogens, highlighting the influence of genetic background and environmental selection on canine immune profiles [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The combination of regulatory (IL-10, NGF-β) and protective (IL-12, IFN-γ) cytokines indicates a finely tuned immune equilibrium that could be harnessed for vaccine development or immunomodulatory therapies aimed at achieving consistent results with reduced immunopathology under endemic exposure.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn an endemic Mediterranean setting, the Fonni dog exhibits a distinct immune signature indicative of tolerance-like immune phenotype under endemic exposure to \u003cem\u003eLeishmania infantum\u003c/em\u003e, rather than susceptibility driven by excessive inflammation. The correlation between pro-inflammatory (IL-6, TNF-α, and MCP-1) and angiogenic (VEGF-α) mediators further suggests coordinated regulation of inflammation and tissue repair.\u003c/p\u003e \u003cp\u003eThe Fonni dog exhibits a distinct cytokine and growth factor signature characterized by elevated IL-10, NGF-β, IFN-γ, TNF-α, and VEGF-α concentrations, supporting a tolerant immune strategy against \u003cem\u003eL. infantum\u003c/em\u003e infection. Fonni dogs appear capable of modulating their immune response in a manner compatible with limited immunopathology under endemic exposure.\u003c/p\u003e \u003cp\u003eOverall, these findings reinforce previous evidence that breed-specific immune adaptations strongly influence the outcome of \u003cem\u003eL. infantum\u003c/em\u003e infection in dogs. Understanding these natural resistance mechanisms in native breeds such as the Fonni dogs may offer valuable models for developing sustainable control strategies for zoonotic leishmaniosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLola Martínez-Sáez: Data curation, methodology, formal analysis, writing – original draft; Raffaella Cocco: Data curation; Luigi Liotta: Data curation, writing-review and editing. Pablo Jesús Marín-García: methodology, formal analysis; Lola Llobat: Writing – review \u0026amp; editing, Writing – original draft, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Universidad Cardenal Herrera, grant numbers INDI25-42, and GIR25-32. Lola Martínez-Sáez is supported by a Predoctoral Contract from the Universidad Cardenal Herrera CEU. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical considerations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures complied with European Directive 2010/63/EU on animal protection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the Universidad Cardenal Herrera CEU (Spain), Universita ` degli Studi di Messina (Italy) and University of Sassari (Italy). This paper is part of the Doctoral Thesis of Lola Martínez-Sáez, and all authors agree.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCosta CHN, Chang K-P, Costa DL, Cunha FVM. From Infection to Death: An Overview of the Pathogenesis of Visceral Leishmaniasis. Pathogens. 2023;12:969. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pathogens12070969\u003c/span\u003e\u003cspan address=\"10.3390/pathogens12070969\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlvar J, V\u0026eacute;lez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis Worldwide and Global Estimates of Its Incidence. PLOS ONE. 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Vet Res Commun. 2023;47:1675\u0026ndash;85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11259-023-10123-z\u003c/span\u003e\u003cspan address=\"10.1007/s11259-023-10123-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cytokines, Fonni dog, immune tolerance, Leishmania, Mediterranean breeds","lastPublishedDoi":"10.21203/rs.3.rs-8367154/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8367154/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCanine leishmaniosis caused by \u003cem\u003eLeishmania infantum\u003c/em\u003e remains a major zoonotic concern in the Mediterranean basin, where native breeds may have evolved adaptive immune mechanisms against infection. This study evaluated cytokine and growth factor profiles in Fonni dogs, a Sardinian native breed, and German Shepherds exposed to \u003cem\u003eL. infantum\u003c/em\u003e and other vector-borne pathogens. Samples were collected as part of routine clinical practice, and no procedures were performed for research purposes. Fifty-nine clinically healthy dogs were analyzed for seropositivity to \u003cem\u003eAnaplasma phagocytophilum\u003c/em\u003e, \u003cem\u003eEhrlichia canis\u003c/em\u003e, \u003cem\u003eLeptospira\u003c/em\u003e spp., \u003cem\u003eLeishmania infantum\u003c/em\u003e, and \u003cem\u003eRickettsia\u003c/em\u003e spp., and for serum levels of eleven cytokines and growth factors using a multiplex immunoassay. Fonni dogs showed significantly higher serum levels of IL-10, NGF-β, IFN-γ, TNF-α, and VEGF-α compared with German Shepherds. \u003cem\u003eL. infantum\u003c/em\u003e and \u003cem\u003eRickettsia\u003c/em\u003e spp. positive animals exhibited elevated IL-10, NGF-β, and TNF-α concentrations. Age influenced several cytokines, with young Fonni dogs displaying the highest IL-10 and NGF-β values, while TNF-α and MCP-1 increased with age. Correlation and principal component analyses revealed distinct clustering between breeds, highlighting coordinated regulation of pro-inflammatory and angiogenic mediators (IL-6, TNF-α, MCP-1, VEGF-α). The elevated values of several cytokines in Fonni dogs suggest a breed-specific immune tolerance-like phenotype consistent with reduced immunopathology under endemic exposure. These data support a breed-associated regulatory/pro-inflammatory immune balance in Fonni dogs, consistent with a tolerance-like immune phenotype under endemic exposure.\u003c/p\u003e","manuscriptTitle":"Breed-specific immune tolerance to Leishmania infantum in a native Mediterranean canine population","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 17:21:41","doi":"10.21203/rs.3.rs-8367154/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-15T14:28:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-15T11:20:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-22T13:58:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79893135047191180357345341683356808651","date":"2025-12-21T18:20:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"195530495851178885201517454575322472598","date":"2025-12-20T11:29:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-19T10:53:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-17T14:00:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-17T04:14:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-12-15T13:48:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c255c370-6537-452c-8831-bda97a5a2519","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:06:04+00:00","versionOfRecord":{"articleIdentity":"rs-8367154","link":"https://doi.org/10.1186/s13071-026-07337-w","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2026-03-09 15:59:08","publishedOnDateReadable":"March 9th, 2026"},"versionCreatedAt":"2025-12-22 17:21:41","video":"","vorDoi":"10.1186/s13071-026-07337-w","vorDoiUrl":"https://doi.org/10.1186/s13071-026-07337-w","workflowStages":[]},"version":"v1","identity":"rs-8367154","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8367154","identity":"rs-8367154","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}