Breed-specific immune regulation under endemic exposure to Leishmania infantum and other vector-borne pathogens in a native Mediterranean canine population

Abstract

Background

Canine leishmaniosis caused by Leishmania infantum remains a major zoonotic concern in the Mediterranean basin, where native breeds may have evolved adaptive immune mechanisms under long-term endemic exposure. The Fonni dog, indigenous to Sardinia, may represent a model of such adaptation. This study aimed to compare cytokine and growth factor profiles between Fonni dogs and German Shepherd dogs exposed to L. infantum and other vector-borne pathogens, to investigate potential breed-associated immune regulatory patterns.

Methods

Fifty-nine clinically healthy dogs (Fonni and German Shepherds) living in endemic areas were included. Serum samples were tested for antibodies against Anaplasma phagocytophilum, Ehrlichia canis, Leptospira spp., Leishmania infantum, and Rickettsia spp. Concentrations of eleven cytokines and growth factors were measured using a multiplex bead-based immunoassay. Statistical analyses evaluated differences between breeds, associations with serological status and age, as well as correlation matrices and principal component analysis to explore clustering patterns among immune mediators.

Results

Fonni dogs showed significantly higher serum concentrations of IL-10, NGF-β, IFN-γ, TNF-α, and VEGF-α compared with German Shepherds. Seropositive dogs for L. infantum and Rickettsia spp. exhibited increased levels of IL-10, NGF-β, and TNF-α. Age influenced cytokine expression, with young Fonni dogs displaying the highest IL-10 and NGF-β values, whereas TNF-α and MCP-1 concentrations increased with age. Correlation and principal component analyses revealed distinct breed-specific clustering, highlighting coordinated regulation of pro-inflammatory and angiogenic mediators, particularly IL-6, TNF-α, MCP-1, and VEGF-α.

Conclusions

The elevated levels of several cytokines and growth factors in Fonni dogs suggest a breed-associated immune phenotype characterized by a regulatory/pro-inflammatory balance consistent with a tolerance-like response under endemic exposure. These findings support the hypothesis that native breeds such as the Fonni dog may have developed adaptive immunological mechanisms that limit immunopathology while maintaining effective responses in areas endemic for canine vector-borne pathogens.

Graphical abstract

Background

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 [1, 2]. Among these pathogens, Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis [3], a disease endemic to Africa, Asia, the Americas, and Europe [4]. Leishmaniasis has been endemic in the Mediterranean basin since antiquity, with historical, paleopathological, and epidemiological evidence supporting long-standing autochthonous transmission of L. infantum sustained by phlebotomine sandflies and canine reservoirs across southern Europe and the wider Mediterranean region [5–9]. Within Europe, visceral leishmaniasis persists in the Mediterranean region, particularly in Spain and Italy [7, 8], where the domestic dog (Canis lupus familiaris) serves as the primary reservoir host [10].

However, dogs in these regions are often exposed not only to L. infantum but also to other pathogens such as Anaplasma phagocytophilum, Ehrlichia canis, Leptospira spp., and Rickettsia spp., which may circulate simultaneously in overlapping ecological niches [11–14]. 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 [15, 16]. The immunopathogenesis of canine leishmaniosis depends largely on the host’s ability to mount a protective Th1 immune response, characterized by interferon (IFN)-γ, interleukin (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 [17–20].

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 [21, 22]. 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 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 [14, 23].

Sardinia represents a unique epidemiological setting, where L. infantum coexists with a high prevalence of other vector-borne pathogens [24, 25]. 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 L. infantum, using the Fonni’s dog as a naturally adapted host and the German Shepherd as a comparative breed.

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 [26].

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. In total, 59 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.

Clinical evaluation was performed by licensed veterinarians at the time of sampling. The absence of clinical signs compatible with canine leishmaniosis was assessed according to the LeishVet international guidelines, including lymphadenomegaly, weight loss, dermatological lesions (alopecia, exfoliative dermatitis, ulcers), ocular abnormalities, epistaxis, onychogryphosis, lethargy, and signs suggestive of renal involvement [27]. Dogs presenting clinical signs compatible with other systemic infectious diseases (e.g., fever, generalized lymphadenomegaly, mucosal pallor, dehydration, jaundice, or altered general condition) were excluded on the basis of routine physical examination. 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’s 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 3 min 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, 10 ml of blood was 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 min. The separated serum was transferred to cryotubes and stored at −80 °C until infection and cytokine determination.

Serological analysis

Serum samples were analyzed to detect antibodies against A. phagocytophilum, E. canis, L. infantum, Leptospira spp., and Rickettsia spp. using pathogen-specific serological methods.

Antibodies against A. phagocytophylum were detected by indirect immunofluorescent assay (IFAT) using antigen-coated slides (Fuller Laboratories), and titers > 1:40 were considered positive. Detection of anti-E. canis antibodies were performed by IFAT following the protocol described by [28]. The presence of antibodies against Leptospira spp. was determined using the microscopic agglutination test (MAT), which is considered the serological reference standard (gold standard) for diagnosis of leptospirosis owing to its high specificity in detecting anti-Leptospira antibodies [29]. The MAT was performed according to the standard procedures recommended by the World Organisation for Animal Health (WOAH, formerly OIE). A panel of live 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), mixed with an equal volume of each live antigen, and incubated at 28–30 °C for 1 h. Agglutination was assessed by dark-field microscopy, and samples showing ≥ 50% agglutination were considered positive. Positive sera were further titrated by serial two-fold dilutions, with titers ≥ 1:100 considered positive.

Anti-L. infantum immunoglobulin G (IgG) antibodies were detected using an in-house IFAT in accordance with the World Organisation for Animal Health (WOAH, formerly OIE) [30], and titers ≥ 1:80 were considered positive. Anti-Rickettsia spp. IgG antibodies were detected using a commercial canine IFAT kit (AffiVET/AffiGEN), 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, nerve growth factor (NGF)-β, IFN-γ, tumor necrosis factor (TNF)-α, MCP-1 (CCL2), vascular endothelial growth factor (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 interaction terms between breed and age, and between breed and infection status, were specified a priori on the basis of biological plausibility and study objectives and were tested irrespective of the statistical significance of the corresponding main effects. 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 labeled 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. The quality of the dataset was assessed to 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 [31].

Results

Study population and serological findings

Of the 59 dogs included in the study, 15 were males (25.4%), and 44 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%). Overall, 27 dogs belonged to the Fonni’s breed (45.8%) and 32 were German Shepherds (54.2%) (Table 1). Serological screening revealed antibodies against Rickettsia spp. in 21 dogs (35.6%, 3 German Shepherd and 18 Fonni’s), Leptospira spp. in 6 dogs (10.2%, 3 German Shepherd and 3 Fonni‘s), E. canis in 3 Fonni’s dogs (5.1%), and L. infantum in 3 dogs (5.1%, 2 Fonni’s and one German Shepherd). 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); a purely descriptive observation reflecting the different concentration ranges of the analytes. After log₁₀ transformation, residual inspection indicated an acceptable model fit for all analytes.

Table 1.

Epidemiological data of dogs 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’s	27 (45.8%)
	German Shepherd	32 (54.2%)
A. phagocytophylum	Positive	0 (0%)
	Negative	59 (100%)
E. canis	Positive (3 F)	3 (5.1%)
	Negative	56 (94.9%)
Leptospira spp.	Positive (3 F + 3 GS)	6 (10.2%)
	Negative	53 (89.8%)
L. infantum	Positive (2 F + 1 GS)	3 (5.1%)
	Negative	56 (94.9%)
Rickettsia spp.	Positive (18 F + 3 GS)	21 (35.6%)
	Negative	38 (64.4%)

F Fonni’s, GS German Shepherd

Table 2.

Mean and standard deviation of all cytokines analyzed

	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

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)

Regarding fixed effects, no statistical differences were found between males and females. Age exerted a significant effect on four cytokines (IL-10, NGF-β, MCP-1, and VEGF-α), whereas breed was associated with significant differences in five cytokines (IL-10, NGF-β, IFN-γ, TNF-α, and VEGF-α; 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’s 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 pg/mL, 22.8 ± 102.4 pg/mL, 4.8 ± 8.8 pg/mL, 19.5 ± 92.4 pg/mL, and 17.4 ± 9.9 pg/mL, respectively). No sex-, age-, or breed-related differences were detected for IL-2, IL-6, IL-8, IL-12, or SCF.

Table 3.

Cytokine serum levels according to the variables

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 ± 37.9a	542.2 ± 295.1	24.4 ± 57.6a	6.0 ± 9.0	12.9 ± 28.8	36.6 ± 26.7a	18.3 ± 17.2a	79.5 ± 55.2
	Young	135.5 ± 177.3	213.4 ± 240.2	1214.2 ± 1226.6	116.3 ± 107.3b	890.4 ± 595.7	149.0 ± 186.8b	15.7 ± 18.0	45.0 ± 88.1	38.7 ± 25.2a	37.4 ± 25.9b	172.3 ± 135.7
	Adult	138.4 ± 325.9	257.9 ± 633.9	1175.3 ± 565.6	26.3 ± 33.8a	907.9 ± 1256.9	52.4 ± 128.5a	5.2 ± 8.8	31.2 ± 103.9	50.8 ± 52.2b	18.4 ± 9.6a	195.1 ± 349.3
	Elder	253.3 ± 449.0	624.2 ± 1269.2	1348.1 ± 597.8	67.87 ± 139.4b	2463.7 ± 4730.2	102.6 ± 283.6b	6.2 ± 8.1	38.8 ± 108.9	96.5 ± 90.2b	37.8 ± 28.2b	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’s	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

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)

Association between infection status and cytokine profiles

Owing 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. 1a) and Rickettsia spp. (Fig. 1b) showed higher serum concentrations of IL-10, NGF-β, and TNF-α compared with seronegative animals (q < 0.05, FDR-adjusted). Owing to the limited number of seropositive dogs, especially for L. infantum, analyses were conducted considering the whole population irrespective of breed, and results should be interpreted with caution.

Fig. 1.

Cytokine concentrations in L. infantum (a) and Rickettsia spp. (b) positive (3 and 21, respectively) and negative (53 and 38, respectively) dogs. Boxplots show the values of cytokines statistically different between the two groups (IL-10, NGF-β, and TNF-α). L. infantum and Rickettsia spp. positive animals presented significantly higher IL-10, NGF-β, and TNF-α values compared with uninfected animals (q < 0.05, Benjamini–Hochberg FDR-adjusted). Results involving L. infantum seropositive dogs should be interpreted with caution owing to the low number of positive animals (n = 3). IL interleukin, NGF nerve growth factor, TNF tumor necrosis factor

Interaction effects between breed, age, and infection status

The pairwise interaction analysis revealed a significant interaction between breed and age for IL-10 and IFN-γ concentrations. For IFN-γ, although no overall age-related main effect was detected, the breed-associated differences varied across age groups, resulting in a significant breed × age interaction. Young Fonni’s 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).

Fig. 2.

IL-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’s breed presented the highest values of these two cytokines (q < 0.05, Benjamini–Hochberg FDR-adjusted). Results involving L. infantum-seropositive dogs should be interpreted with caution owing to the low number of positive animals (n = 3). IL interleukin, IFN interferon. Number of animals included by category: 27 Fonni’s (1 elder, 6 adults, 10 puppies, and 10 young) and 24 German Shepherd (3 elders, 15 adults, 4 puppies, and 2 young)

Fig. 3.

IL-10 (a), NGF-β (b), and TNF-α (c) serum levels according to age group in infected (positive) and uninfected (negative) Rickettsia spp. dogs. Boxplots show the values of cytokines statistically different between the groups. Elder infected dogs presented the highest values of these three cytokines (q < 0.05, Benjamini–Hochberg FDR-adjusted). IL interleukin, NGF nerve growth factor, TNF tumor necrosis factor. Number of animals included by category: 20 infected (2 elders, 6 adults, 3 puppies, and 9 young) and 24 uninfected (2 elders, 15 adults, 11 puppies, and 3 young)

Cytokine correlation analysis

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) is 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.

Fig. 4.

Spearman 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 (q-value < 0.05)

Multivariate analysis of cytokine profiles

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, and SCF), whereas PC2 was associated with regulatory and modulatory responses (NGF-β, IL-10, TNF-α, and IFN-γ). Visualization of PCA scores indicated partial separation according to breed and L. infantum serological status (Fig. 5a), with Fonni’s dogs clustering toward higher PC2 values (Fig. 5b).

Fig. 5.

Principal component analysis (PCA) of cytokine and immune factor profiles. PCA was conducted using standardized concentrations of 11 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 L. infantum infection status, and (b) cluster according to dog breed. Given the low number of L. infantum-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

Discussion

This study provides evidence that prolonged historical exposure, occurring over centuries to millennia under Mediterranean endemic conditions, to vector-borne pathogens can shape breed-specific immune regulation in dogs, with the Fonni’s breed displaying a profile consistent with infection tolerance rather than inflammatory susceptibility. This observation does not imply direct genetic co-evolution over a defined evolutionary timescale, but rather long-standing exposure across multiple generations under stable endemic pressure. 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’s dogs displayed significantly higher serum levels of IL-10, NGF-β, IFN-γ, TNF-α, and VEGF-α compared with 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’s breed, which is consistent with previous reports suggesting that Mediterranean native breeds exposed to long-standing endemic vector-borne pathogens may display immune regulatory patterns compatible with partial resistance or tolerance to L. infantum infection [32]. However, German Shepherds originated in a temperate European environment with different ecological and vector-borne disease pressure and therefore should be regarded as a comparative population rather than an evolutionary control. Consequently, the observed differences may reflect contrasting historical exposure to vector-borne pathogens rather than exclusively intrinsic breed-specific adaptations.

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’s dogs displayed the highest IL-10 and NGF-β levels, whereas adults and elders showed increased MCP-1. These findings agreed with studies reporting Th2-polarized responses in immature immune systems [33–35] and inflammation-associated cytokine increases with aging [36, 37]. 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 [38–40]. Considering these findings, both Leishmania and Rickettsia spp. seropositives were associated with increased IL-10, NGF-β, and TNF-α concentrations, suggesting overlapping immune regulatory responses to chronic or repeated vector-borne pathogen exposure. However, L. infantum remains epidemiologically and biologically distinct as a persistent intracellular protozoan and the main zoonotic pathogen in the Mediterranean basin, whereas Rickettsia spp. are associated with transient bacterial infections. Thus, similarities probably result from common regulatory pathways rather than the same immunopathogenic processes.

The constitutively elevated IL-10 and NGF-β levels observed in Fonni’s dogs, independent of infection status, suggest a preexisting 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 [18, 41, 42]. NGF-β is increasingly recognized as a regulator of macrophage oxidative activity and immune cell survival [43, 44]. Experimental models show that NGF-β enhances macrophage hydrogen peroxide production and inhibits L. donovani replication [45], 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 [46–48]. Fonni’s dogs also exhibited higher IFN-γ levels than German Shepherds, particularly in young animals. This cytokine is central to Th1 polarization and macrophage activation [49–52]. Their co-existence with elevated IL-10 suggests a balanced immune regulation that can control intracellular pathogens while minimizing collateral tissue damage. Such an equilibrium has been proposed in other naturally resistant breeds such as the Ibizan hound [32].

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 remodeling. 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 [53, 54]. Fonni’s 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’s from German Shepherds along principal components dominated by pro-inflammatory and anti-inflammatory cytokines. This statistical clustering supports the hypothesis that Fonni’s dogs maintain a distinct immune signature, probably shaped by long-term exposure to Sardinian eco-epidemiological conditions [24, 55, 56].

Taken together, the Fonni’s 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 [22, 32, 57]. 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

Fonni’s 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’s 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 Fonni’s dogs may offer valuable models for developing sustainable control strategies for zoonotic leishmaniosis, including the identification of immune biomarkers associated with tolerance, the design of immunomodulatory or vaccine approaches aimed at limiting immunopathology rather than inducing sterilizing immunity, and the use of naturally tolerant breeds as epidemiological models to inform integrated disease control in endemic settings.

Author contributions

Lola Martínez-Sáez: data curation, methodology, formal analysis, and 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 and editing, writing—original draft, supervision, project administration, investigation, funding acquisition, formal analysis, and conceptualization.

Funding

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.

Data availability

Data supporting the main conclusions of this study are included in the manuscript.

Declarations

Ethics approval and consent to participate

All procedures complied with European Directive 2010/63/EU on animal protection.

Competing interests

The authors declare no competing interests.