Splenic plasticity at the arid limit: dehydration is associated with microanatomical remodelling and B-cell depletion in the dromedary camel (Camelus dromedarius)

Abstract

Background

The dromedary camel (Camelus dromedarius) is the exemplar of mammalian adaptation to hyper-arid environments, possessing specialized physiological mechanisms to tolerate extreme osmotic stress and hemoconcentration. While renal and thermoregulatory adaptations are well-characterized, the costs of these survival strategies on immune microanatomy remain poorly defined. Structural remodeling of lymphoid organs may accompany hydric and metabolic stress in xeric specialists, but whether such changes represent adaptive reallocation or a nonspecific consequence of physiological stress remains uncertain. This study investigates the plasticity of the splenic marginal zone (MZ), a critical reservoir for innate-like B cells, under naturally occurring dehydration gradients.

Methods

We conducted a cross-sectional morphophysiological study on C. dromedarius (n = 100) in the hyper-arid El Oued region of the Algerian Sahara. Hydration status was defined via serum osmolality and hematocrit. We utilized quantitative histomorphometry and immunohistochemistry to assess MZ thickness and B-cell density. Multivariable linear regression was employed to control for covariates (age, sex, body condition). We applied statistical mediation analysis to examine whether MZ remodeling statistically accounted for part of the association between dehydration and B-cell density.

Results

Dehydrated camels exhibited significant splenic plasticity, characterized by a marked reduction in MZ thickness (157.9 ± 10.8 μm vs. 183.1 ± 9.6 μm in hydrated controls; p < 0.001) and a concomitant 24% decline in B-cell density. MZ atrophy was strongly correlated with systemic hydration markers (serum osmolality: r = -0.72). Mediation modeling indicated that 59% of the dehydration–B-cell density association was statistically accounted for by the MZ-thickness pathway (ACME: -196.2 cells/mm²). Furthermore, animals from the hyper-arid Sahara micro-region displayed greater MZ contraction than those from the transitional Sahel, suggesting environmental modulation of splenic architecture.

Conclusions

These findings demonstrate marked dehydration-associated plasticity of the splenic MZ in the dromedary camel. The concurrent reduction in MZ thickness and B-cell density, together with their statistical association, identifies a previously undercharacterized relationship between systemic hydration status and splenic microanatomy. The present data do not establish that lymphoid remodeling is itself beneficial, adaptive, or specific to water deprivation; it may also reflect a broader physiological response to metabolic, endocrine, hemodynamic, nutritional, or other stressors. The findings therefore provide a basis for further investigation of the mechanisms and functional significance of splenic remodeling during dehydration.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s40850-026-00284-0.

Introduction

Severe dehydration imposes simultaneous challenges on water balance, thermoregulation, circulation, and energy metabolism [1, 2], and these systemic changes can influence immune function [3, 4]. Although immune down-regulation has been proposed as one component of resource allocation during physiological stress [3, 4], stress-associated changes in lymphoid organs may also arise as nonspecific consequences of endocrine, metabolic, nutritional, or inflammatory perturbation. Chronic stress, for example, has been associated with structural changes in splenic lymphoid tissue in experimental mammals [5]. While the renal and cardiovascular adaptations of the dromedary camel (Camelus dromedarius) are well documented [6, 7], considerably less is known about how severe dehydration is reflected at the level of splenic microarchitecture.

The dromedary camel (Camelus dromedarius) represents a highly specialized mammalian model of physiological adaptation to the Saharan ecosystem, possessing specialized mechanisms to tolerate severe hemoconcentration and body water losses exceeding 25% of total mass [6, 7]. Central to this resilience is the spleen, an organ that serves a dual function in this species: acting as a capacious reservoir for erythrocytes to support oxygen transport during stress, and functioning as a primary site for immune surveillance [8–10]. The splenic marginal zone (MZ) is of particular functional significance; it forms the interface between the systemic circulation and the adaptive immune system, housing specialized MZ B-cells responsible for the rapid neutralization of blood-borne pathogens [11, 12]. Because the structural maintenance of the MZ is heavily dependent on specific microvascular flow patterns and stromal signals [13], this compartment is theoretically vulnerable to the rheological shifts associated with dehydration-induced blood viscosity.

Despite the dromedary’s status as a model organism for desert physiology, no study has yet quantified the relationship between systemic hydration status and splenic microarchitecture in free-ranging populations. Existing literature has largely remained bifurcated, describing either gross anatomical features or systemic hematological parameters in isolation, without integrating organ-level morphometry with physiological stressors [14, 15]. Consequently, it remains unknown whether dehydration-associated splenic remodeling represents a dehydration-specific physiological response, a broader stress response, or some combination of these processes. This absence of quantitative, organ-level data limits our understanding of how dehydration is associated with splenic microarchitecture and immune cellularity in naturally exposed animals.

To address this gap, the present study investigates the relationship between systemic hydration status and splenic microarchitecture in dromedary camels from southeastern Algeria. Integrating multivariable modeling and statistical mediation analysis, we examine whether dehydration status is associated with structural contraction of the splenic MZ and reduced B-cell density, and whether variation in MZ thickness statistically accounts for part of the observed association between dehydration and B-cell density. We use the mediation model as a hypothesis-generating framework rather than as proof of a temporal causal pathway. In addition, we assess whether the magnitude of the observed remodeling differs between hyper-arid Saharan and transitional steppe populations.

Materials and methods

Study area and climatology

The study was conducted in two distinct bioclimatic zones in southeastern Algeria to capture ecological heterogeneity in water availability and heat stress. The primary sampling site was El Oued (33°22′N, 6°52′E), located in the hyper-arid Saharan biome (Grand Erg Oriental). This region is characterized by a hot desert climate (Köppen BWh), with summer maxima frequently exceeding 45 °C, annual precipitation < 80 mm, and scarce halophytic vegetation [16].

To assess regional plasticity, comparative sampling was conducted in Bir El Ater (34°45′N, 8°03′E), located approximately 200 km north in the steppe-transition zone (Pre-Saharan/Sahelian fringe). This area exhibits a semi-arid climate (Köppen BSk), characterized by relatively lower mean annual temperatures and marginally higher vegetative biomass compared to the deep Sahara [17]. This gradient allowed for the evaluation of regional differences in splenic remodeling across distinct arid and semi-arid environmental conditions. The geographic and topographic distinctness of this transect is illustrated in Fig. 1.

Fig. 1.

Topographic and bioclimatic gradient of the study area. Map illustrating the sampling transect (dashed line) across the aridity threshold in southeastern Algeria. Points mark the distinct transition from the hyper-arid Grand Erg Oriental (El Oued) to the semi-arid Nemencha Steppe (Bir El Ater). Background coloration represents the elevation gradient (meters)

Animals and sampling design

A total of 100 adult dromedary camels (Camelus dromedarius) were included in this cross-sectional study (Fig. 2). Animals were selected from municipal abattoirs in the respective regions between 2023 and 2025, with sampling distributed across both hot (dry) and cooler seasons to capture natural environmental variance. Animals were typically sourced from local regional herds, minimizing prolonged pre-slaughter transport durations, and were subjected to standard lairage rest periods.

Fig. 2.

Integrated morphophysiological framework linking systemic hydration status to splenic microanatomy and cellularity

To minimize the confounding effects of subclinical infections and parasite burdens, animals were selected only after passing rigorous ante-mortem and post-mortem inspections by official state veterinarians. Inclusion criteria were: clinically healthy adults (aged 4–15 years) and non-pregnant/non-lactating females. Animals were strictly excluded if they exhibited generalized lymphadenopathy, visceral parasitic cysts (e.g., hydatidosis), signs of trypanosomiasis or tuberculosis, or any pathological lesions warranting partial or total carcass condemnation.

Metadata recorded for each individual included age (dentition estimation), sex, origin (Sahara/El Oued vs. Steppe/Bir El Ater), and Body Condition Score (BCS). BCS was assessed on a 5-point scale validated for dromedaries, where 1 indicates emaciation and 5 indicates obesity [18].

Physiological hydration assessment

Ante-mortem blood samples were collected via jugular venipuncture into plain and EDTA-coated vacutainers. Serum was separated by centrifugation at 1500 × g for 10 min. Hydration status (the primary exposure variable) was dichotomized into “Hydrated” (n = 58) and “Dehydrated” (n = 42) based on established physiological thresholds for camelids [7, 19]:

Serum osmolality: Measured via freezing-point depression osmometry [20], Consistent with established physiological baselines for Camelus dromedarius, where euhydrated serum osmolality typically ranges between 290 and 300 mOsm/kg, animals exhibiting hyperosmolality > 310 mOsm/kg were classified as dehydrated [7, 21–23].

Hematocrit (Packed cell volume): Determined via micro-capillary centrifugation. Elevated PCV values consistent with hemoconcentration (> 35–40% depending on age/sex norms) corroborated the osmolality classification.

Histology and immunohistochemistry (IHC)

Spleen samples were harvested within 45 min of slaughter to minimize autolytic changes. To ensure anatomical consistency across all 100 animals, tissue blocks (~ 1 cm³) were systematically excised from the mid-parietal surface of the spleen, equidistant from the cranial and caudal poles. To maintain a standardized depth and orientation, sections were cut perpendicularly to the capsule, ensuring each block captured the full architectural continuum spanning the external capsule, subcapsular red pulp, and deep white pulp.

Tissue Processing: Samples were fixed in 10% neutral buffered formalin (NBF) for 24–48 h, dehydrated through graded ethanol, cleared in xylene, and embedded in paraffin wax.

Staining: Serial Section  (4–5 μm) were cut using a rotary microtome. Routine Hematoxylin and Eosin (H&E) staining was performed for morphological assessment.

Immunophenotyping: To quantify B-cell density, immunohistochemistry was performed using a rabbit anti-CD20 polyclonal antibody. Species cross-reactivity and specificity for Camelus spp. were previously validated [14], and further confirmed internally in our study by the localization of positive staining exclusively to the expected B-cell follicular and marginal compartments. Antigen retrieval was achieved via heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0). Detection utilized a horseradish peroxidase (HRP) polymer system with 3,3’-diaminobenzidine (DAB) chromogen, counterstained with Mayer’s hematoxylin. Methodological controls included the omission of the primary antibody (negative control) to assess non-specific background staining, and the use of standard camel lymph node tissue as a positive control.

Digital image analysis and morphometry

Histomicrographs were digitized using a high-resolution optic microscope equipped with camera. Morphometric analysis was conducted using QuPath (v0.4.3) and ImageJ/FIJI software. All digital slides were numerically coded to ensure investigators were strictly blinded to both the hydration status and eco-regional origin of the animals during analysis.

MZ thickness: Defined as the perpendicular distance from the outer boundary of the white pulp (periarteriolar lymphoid sheath/follicle) to the inner limit of the red pulp. To prevent selection bias, a digital grid overlay was utilized to randomly select 5 distinct, non-overlapping follicles per animal. Measurements were taken at 4 equidistant orthogonal points per selected follicle and averaged to generate a mean MZ thickness (µm). To validate measurement reproducibility, a subset of 20 randomly selected slides was independently evaluated by a second blinded investigator. Inter-observer reliability was assessed using the intraclass correlation coefficient (ICC), demonstrating excellent agreement (ICC = 0.91).

B-Cell density: The Region of Interest (ROI) was defined strictly within the boundaries of the MZ. To account for intra-slide variability and heterogeneous cellular distribution, a minimum of five distinct, non-overlapping ROIs were analyzed per slide and averaged. CD20-positive cells were quantified using QuPath’s built-in “Positive Cell Detection” algorithm. Prior to quantification, stain vectors (Hematoxylin and DAB) were calibrated utilizing the “Estimate Stain Vectors” function to ensure accurate optical density deconvolution. A standardized DAB intensity threshold was empirically established against the negative controls to strictly exclude false-positive background chromogen deposition. Final cell counts were normalized to the ROI surface area and expressed as cells/mm².

Statistical analysis

Data analysis was performed using R Statistical Software (v4.3.1).

Multivariable modeling

To assess the impact of hydration status while controlling for potential confounders, we utilized General Linear Models (GLM). Hydration status (Dehydrated vs. Hydrated) was entered as the primary fixed effect, with Age, Sex, Region, and Body Condition Score (BCS) included as covariates. Multicollinearity was assessed by calculating the Variance Inflation Factor (VIF); all VIF values were < 2.0, indicating no problematic collinearity.

Model diagnostics and effect sizes

Prior to finalizing the GLMs, parametric assumptions were rigorously verified. Residual normality was confirmed via Shapiro-Wilk tests and visual inspection of Q-Q plots, and homoscedasticity was verified using the Breusch-Pagan test. To quantify the magnitude of associations beyond simple p-values, standardized regression coefficients (β) and Cohen’s d effect sizes were calculated. To account for multiple testing across our primary physiological and morphometric outcomes, p-values were adjusted using the Benjamini-Hochberg False Discovery Rate (FDR) procedure. Finally, a post-hoc power analysis indicated that our sample size of N = 100 provided > 85% statistical power to detect the observed moderate-to-large effect sizes at an α = 0.05 significance level. Furthermore, a sensitivity analysis was conducted treating serum osmolality as a continuous predictor to ensure robustness of the morphological associations [24].

Mediation analysis

To examine whether MZ thickness statistically accounted for part of the association between dehydration status and B-cell density, we performed a mediation analysis using the mediation package [25]. Dehydration status was defined as the exposure (X), MZ thickness as the mediator (M), and B-cell density as the outcome (Y). The Average Causal Mediation Effect (ACME) and Average Direct Effect (ADE) were estimated using non-parametric bootstrap resampling (5,000 iterations). Significance was defined as p < 0.05. The mediation analysis was interpreted as a statistical decomposition of association rather than definitive evidence of a biological causal pathway, because the cross-sectional design does not establish temporal ordering and the assumptions required for causal mediation cannot be fully verified.

Results

Physiological validation: systemic hemoconcentration in the dehydrated dromedary

Systemic biomarkers confirmed a distinct state of hemoconcentration in the dehydrated group (Table 1). Dehydrated animals exhibited a marked elevation in serum osmolality (mean increase of + 49.7 mOsm/kg, p < 0.001) and hematocrit (+ 11.6%, p < 0.001). A strong positive correlation between these two markers (r = 0.81, p < 0.001) validated the use of packed cell volume as a reliable proxy for osmotic stress in this free-ranging population (Fig. 3).

Table 1.

Demographic and physiological profile of the study population stratified by hydration status

Parameter	Hydrated (n = 58)	Dehydrated (n = 42)	Effect size (Cohen’s d)	p-value
Age (years)	8.1 ± 2.8	8.5 ± 3.1	-	0.48
Sex (% Female)	52%	48%	-	0.67
Serum osmolality (mOsm/kg)	285.4 ± 12.3	335.1 ± 18.7	3.25	< 0.001
Hematocrit (%)	30.2 ± 3.5	41.8 ± 4.2	3.05	< 0.001
MZ Thickness (µm)	183.1 ± 9.6	157.9 ± 10.8	2.49	< 0.001
B-Cell ensity (cells/mm²)	528.8 ± 103.4	400.4 ± 95.1	1.28	0.004

Values are presented as Mean ± Standard Deviation (SD)

Fig. 3.

Physiological validation of dehydration status via systemic biomarkers. Scatter plot displaying the relationship between serum osmolality (mOsm/kg) and hematocrit (%) in the study population

Morphometric plasticity: dehydration is associated with significant atrophy of the splenic MZ

Quantitative histomorphometry revealed a profound structural Remodelling of the spleen associated with hydric stress. The MZ—the critical interface for blood-borne antigen capture—underwent significant atrophy, with mean thickness reduced by 13.7% in dehydrated animals relative to hydrated controls (t = 12.4, p < 0.001). Sensitivity analysis utilizing continuous serum osmolality confirmed this relationship, demonstrating a significant negative linear association with MZ thickness (β = -0.68, p < 0.001).

The distributional analysis highlights a uniform phenotypic shift. The dehydrated cohort displayed a clear downward shift in median thickness (Fig. 4). Furthermore, the ridge density estimation in demonstrated a “bimodal compression,” characterized by a shift toward a narrower, minimal structural baseline. This suggests that dehydration may impose a physiological constraint that overrides natural morphological variation.

Fig. 4.

Quantitative evidence of dehydration-induced splenic atrophy. Raincloud plot illustrating the structural remodelling of the Marginal Zone (MZ) under hydric stress. The visualization combines a half-violin plot (density distribution), a box-and-whisker plot (median and interquartile range), and jittered raw data points to show the full data structure

Cellular dynamics: structural contraction limits the MZ B-Cell niche

Parallel to the microanatomical atrophy Fig. 5A–B, immunohistochemical analysis demonstrated a significant depletion of the resident immune population Fig. 5C–I. B-cell density within the MZ was suppressed by 24.2% in the dehydrated cohort (400.4 ± 95.1 cells/mm²) compared to the hydrated baseline (528.8 ± 103.4 cells/mm²; p = 0.004).

Fig. 5.

Dehydration significantly reduces B-cell population and alters splenic marginal zone architecture. (A, B) Hematoxylin and eosin (H&E) stained sections of the spleen showing a well-defined marginal zone in the hydrated camel (A, width indicated by continuous double-headed arrows) and a contracted, less distinct zone in the dehydrated camel (B, width indicated by discontinuous double-headed arrows). (C, D) Low-magnification immunohistochemistry (IHC) for B-cells (brown), confirming a dense population in the hydrated state (C) and a marked reduction in density in the dehydrated state (D). (E, F) the high-magnification IHC, further detailing the reduced B-cell density in the dehydrated sample (F) compared to the hydrated control (E). (G, H) Immunofluorescence visualization (yellow) of B-cells, consistent with IHC findings for the two groups. (I) Quantitative analysis comparing total and IHC + B-cell counts, showing a significant decrease in the proportion of positive B-cells in the dehydrated condition. Scale bars = 300 μm (A, B); 100 μm (C, D); 25 μm (E, F)

Regression modeling indicates that this cellular loss is structurally constrained. As illustrated in Fig. 6, MZ thickness was a robust positive predictor of B-cell density (R² = 0.59, p < 0.001), suggesting that the physical collapse of the MZ compartment directly limits the available niche space required for B-lymphocyte retention.

Fig. 6.

Multivariate integration of the dehydration-immunology interaction. Weighted scatter plot visualization mapping the triadic interaction between systemic osmotic stress (serum osmolality), splenic microarchitecture (MZ thickness), and immune cellularity (B-cell density)

The dehydration–immunology interaction: systemic osmolality predicts splenic microarchitecture

Multivariate correlation analysis revealed a coherent “stress syndrome” linking systemic fluid balance to splenic integrity. As visualized in the correlation matrix (Fig. 7), MZ thickness exhibited strong, significant negative correlations with the primary indices of dehydration: Serum osmolality (r = -0.72) and hematocrit (r = -0.69). Conversely, a positive correlation was observed between MZ thickness and BCS (r = 0.63), indicating that energetic reserves may buffer against stress-induced atrophy. The absence of a significant correlation with Age (r = -0.33) suggests that this remodelling is an acute physiological response rather than a senescent decline.

Fig. 7.

Multivariate correlation matrix of physiological and splenic parameters. Pearson correlation heatmap visualizing the pairwise relationships between systemic variables (Age, Body Condition Score, Osmolality, Hematocrit) and splenic microanatomy (Marginal Zone Thickness, B-Cell Density). The color gradient represents the strength and direction of the correlation coefficient (r), ranging from dark red (positive) to dark blue (negative)

Eco-geographic modulation: regional aridity amplifies splenic remodelling

The magnitude of splenic plasticity was modulated by the ecological severity of the habitat. Camels originating from the arid Saharan micro-region (El Oued) displayed significantly greater MZ contraction compared to those from the transitional steppe zone (Bir El Ater).

This finding, visualized in Fig. 8, The significant regional difference indicates that environmental context may modulate splenic architecture. However, regional contrasts cannot by themselves establish acclimatization or evolutionary adaptation, because lifelong nutritional history, management conditions, and other ecological differences may contribute to the observed baseline differences.

Fig. 8.

Eco-geographic modulation of splenic plasticity across aridity gradients. Comparative box-and-whisker plots illustrating the influence of regional environmental severity on Marginal Zone (MZ) thickness

Statistical mediation: MZ thickness accounts for part of the dehydration–B-cell association

To test the mechanistic hypothesis that structural remodelling predicts cellular loss, a statistical mediation analysis was performed (Table 2).

Table 2.

Statistical mediation analysis of dehydration on B-Cell density

Effect component	Estimate (Cells/mm²)	95% CI Lower	95% CI Upper	p-value
ACME (Indirect Effect)	-196.2	-254.1	-145.8	< 0.001
ADE (Direct Effect)	-136.3	-201.5	-70.2	0.004
Total effect	-332.5	-410.2	-255.8	< 0.001
Proportion Mediated	0.59 (59%)	0.42	0.76	< 0.001

Estimates generated via 5,000 non-parametric bootstrap simulations

The analysis decomposed the total association between dehydration status and B-cell density (-332.5 cells/mm²) into model-estimated indirect and direct components. The Average Causal Mediation Effect (ACME), representing the statistical pathway from dehydration status through MZ thickness to B-cell density, was estimated at -196.2 cells/mm² (p < 0.001) (Fig. 9). The proportion mediated was estimated at 59%. This value indicates that, within the specified statistical model, 59% of the observed association was attributed to the MZ-thickness pathway. It should not be interpreted as proof that dehydration physically collapses the MZ, that MZ collapse causes B-cell loss, or that the remodeling is adaptive.

Fig. 9.

Directed acyclic graph (DAG) and statistical mediation path diagram illustrating the hypothesized architecture of B-cell loss. The model decomposes the total association of dehydration into direct physiological pathways (ADE) versus indirect structural mediation (ACME), with path strengths quantified via standardized regression coefficients (β)

Quantifying the environmental cost

The interaction between regional aridity and physiological stress (Table 3.) was significant, supporting the hypothesis of an eco-geographic interaction (p < 0.001) regarding splenic architecture. While dehydration induced atrophy in both populations, the magnitude of this collapse was disproportionately severe in the Saharan cohort. Animals from the -arid El Oued region exhibited a 15.9% reduction in MZ thickness (from 176.4 µm to 148.2 µm) compared to a milder 10.6% reduction in the semi-arid Steppe population. Crucially, the ‘Baseline’ column in Table 3 demonstrates that Saharan camels possess a significantly thinner MZ even when hydrated (176.4 ± 8.5 μm vs. 188.5 ± 10.2 μm). We hypothesize this “thinner baseline phenotype” may reflect chronic eco-physiological acclimatization to a resource-scarce environment, though differences in lifelong nutritional history between the regions cannot be excluded as a primary driver.

Table 3.

Eco-geographic modulation of physiometric parameters. Stratified comparison of physiological and morphometric indices between the hyper-arid Saharan (El Oued) and semi-arid Steppe (Bir El Ater) populations under distinct hydration states

Parameter	Region	Hydrated (Baseline)	Dehydrated (Stress)	Δ Change (%)	Interaction P-value†
Serum Osmolality	Arid (Sahara)	288.5 ± 14.1	341.2 ± 19.5	+ 18.2%	0.032*
(mOsm/kg)	Semi-Arid (Steppe)	281.2 ± 10.4	324.6 ± 15.2	+ 15.4%
Hematocrit	Arid (Sahara)	31.4 ± 3.8	44.1 ± 4.5	+ 40.4%	0.091
(%)	Semi-Arid (Steppe)	29.5 ± 3.1	39.2 ± 3.9	+ 32.8%
MZ Thickness	Arid (Sahara)	176.4 ± 8.5	148.2 ± 9.4	-15.9%	< 0.001***
(µm)	Semi-Arid (Steppe)	188.5 ± 10.2	168.4 ± 11.1	-10.6%
B-Cell Density	Arid (Sahara)	495.2 ± 98.4	365.4 ± 88.6	-26.2%	0.015*
(cells/mm²)	Semi-Arid (Steppe)	562.1 ± 110.5	445.8 ± 92.3	-20.6%

Values are presented as Mean ± Standard Deviation (SD)

Δ Change: Represents the relative percentage difference calculated as ((Dehydrated - Hydrated) / Hydrated) × 100

† Interaction P-value: Derived from a Two-Way ANOVA (Factors: Region, Hydration). This value tests the Region × Hydration interaction term; a significant result (p < 0.05) indicates that the physiological impact of dehydration is significantly different (magnitude of change) between the Arid and Semi-Arid populations

Significance levels: * p < 0.05; ** p < 0.01; *** p < 0.001. (p > 0.05)

Discussion

Dehydration-associated splenic remodeling

The present study identifies a robust association between systemic dehydration markers and splenic microanatomical remodeling in Camelus dromedarius. Dehydrated animals showed a substantial reduction in MZ thickness together with lower MZ B-cell density, and these changes remained associated with serum osmolality after multivariable adjustment. These findings extend previous observations that dehydration in dromedaries is accompanied by systemic physiological stress and measurable tissue remodeling [26]. Importantly, the present findings should not be interpreted as demonstrating an ‘immuno-hydric trade-off’ in the evolutionary or functional sense. A reduction in lymphoid tissue is not, on the basis of the present data, demonstrably beneficial during water deprivation. The observed pattern may instead represent physiological remodeling secondary to hemoconcentration, altered perfusion, endocrine stress, altered energy balance, nutritional status, or other concurrent stressors. The principal contribution of this study is therefore the identification and quantification of dehydration-associated splenic remodeling, rather than demonstration of an adaptive benefit of lymphoid depletion.

Hemodynamic constraints and structural remodelling

The high correlation observed between systemic hemoconcentration (hematocrit/osmolality) and MZ thickness is consistent with a possible hemodynamic contribution to the observed remodeling. The dromedary spleen is unique among ungulates for its extensive subcapsular sinus system and rapid blood-storage capacity [8, 9, 27]. As dehydration progresses, blood viscosity increases exponentially due to plasma volume contraction; a severe hemodynamic shift comparable to the systemic fluid-loss cascades observed in acute veterinary inflammatory pathologies [28] and extreme environmental heat stress. We hypothesize that the observed MZ thinning may partly reflect altered vascular dynamics associated with hemoconcentration; however, this mechanism remains untested [1]. However, we must explicitly note that this hemodynamic mechanism remains speculative. Because our current methodology did not include specific microvascular endothelial staining (CD31 or von Willebrand factor), direct quantification of sinusoidal diameters, or in vivo blood flow measurements, this remains an untested hypothesis. If such a mechanism occurs, reducing perfusion to the intricate marginal sinusoids could theoretically allow the camel to shunt blood volume to vital central organs, thereby limiting the “metabolic overhead” of maintaining the expanded stromal network required for lymphocyte retention [13]. Although such a redistribution of physiological resources is biologically plausible, the present study provides no direct evidence that MZ remodeling reduces whole-body water loss, improves hemodynamic stability, or enhances survival during dehydration [29].

Our observation of profound hemoconcentration aligns with recent longitudinal studies on dromedary dehydration. Ali et al. [26] and Faraz et al. [30] similarly reported significant, yet reversible, elevations in hematocrit and red blood cell indices during water restriction. However, the unique rheological properties of camel erythrocytes, which can undergo significant shrinkage during dehydration and swelling during rehydration without lysis [31], likely allow for continued, albeit altered, perfusion through the splenic red pulp even as the marginal zone architecture collapses [23].

Statistical association between MZ remodeling and B-cell density

The mediation analysis provides additional evidence of strong structural–cellular coupling within the MZ. Approximately 59% of the dehydration–B-cell density association was statistically accounted for by the MZ-thickness pathway in the specified model (Table 2). We therefore interpret the result as hypothesis-generating evidence of structural–cellular dependence rather than as proof of a causal pathway [32]. In particular, the data do not establish that dehydration first causes MZ contraction and that this contraction subsequently causes B-cell displacement or apoptosis [11, 12]. Alternative explanations remain possible, including parallel effects of metabolic, endocrine, nutritional, inflammatory, or hemodynamic stress on both splenic structure and cellularity. The residual Direct Effect (ADE) of dehydration likely reflects the concurrent impact of systemic endocrine stress axes (HPA axis activation) on lymphocyte viability, consistent with observations in heat-stressed avian and reptilian models [4, 33].

The reduction in B-cell density observed here, presents a nuanced counterpoint to the previous reports of generalized lymphocytic hyperplasia in the dehydrated camel spleen [26], I which n the authors noted an overall increase in white pulp cellularity, potentially indicative of a systemic stress response, while our data reveals that the marginal zone specifically undergoes atrophy. This suggests that splenic responses to dehydration may be compartment-specific rather than uniformly suppressive, even while deep cortical regions may exhibit hyperplasia. This distinct microanatomical collapse parallels the parenchymal remodelling and vacuolization observed in other mammalian models of dehydration [34], reinforcing the hypothesis that the MZ is uniquely vulnerable to hemodynamic shifts compared to the deeper periarteriolar lymphoid sheaths.

Regional modulation of splenic remodeling

The significant divergence in splenic morphology between the arid El Oued (Sahara) and the transitional Bir El Ater (Steppe) populations underscores the phenotypic plasticity of the dromedary immune system. Animals inhabiting the deep Sahara exhibited a “thinner baseline” MZ even after controlling for acute hydration status. We hypothesize that chronic exposure to extreme heat load and water scarcity may drive a functional acclimatization of splenic architecture. While this could theoretically represent a developmental response to chronic environmental stress, as shown in other species [5], it is equally plausible that alternative factors; most notably, chronic disparities in forage quality and nutritional history between the hyper-arid Sahara and the semi-arid Steppe; contribute to this thinner baseline. Further longitudinal and genetic studies would be required to determine whether these regional differences reflect developmental programming, long-term acclimatization, or persistent differences in environmental and nutritional exposure [2].

Implications for host defense

The functional consequence of this remodelling is a potential transient vulnerability to blood-borne pathogens. The MZ is the primary site for the capture of encapsulated bacteria and the initiation of T-independent antibody responses [14]. The lower B-cell density observed in dehydrated animals could be associated with altered splenic immune capacity, but no functional immune assay was performed in the present study. We cannot therefore determine whether the observed reduction is transient, detrimental, neutral, or beneficial during dehydration. Furthermore, the positive correlation with Body Condition Score suggests that energetic status may influence the magnitude of dehydration-associated splenic remodeling; however, the present data do not establish whether nutritional status directly buffers or modifies this response.

Statistical mediation and directionality

While the mediation model estimated a significant indirect component and a proportion mediated of approximately 59%, these estimates should not be interpreted as absolute mechanistic evidence. Formal causal mediation relies on assumptions that cannot be fully verified in a cross-sectional observational design. Because dehydration status, MZ thickness, and B-cell density were measured at a single time point [35], the temporal direction of these relationships cannot be established empirically. In particular, the present data cannot distinguish a sequential pathway in which dehydration-associated remodeling precedes B-cell loss from parallel effects of metabolic, endocrine, nutritional, inflammatory, or hemodynamic stress on both structure and cellularity [36]. The observed association between MZ thickness and B-cell density nonetheless provides a biologically relevant basis for future longitudinal and mechanistic investigation [37].

Limitations and future directions

This study has limitations inherent to its design. First, the use of cross-sectional data from opportunistic abattoir surveys precludes the establishment of definitive temporal causality; we cannot empirically rule out reverse causation.

A further limitation is that the present design cannot determine whether the observed lymphoid remodeling is specific to dehydration or represents a broader response to physiological stress. Because cortisol and other endocrine stress markers were not measured, and detailed information on nutritional history, inflammatory status, and exact pre-slaughter stress exposure was unavailable, residual confounding by concurrent metabolic or stress-related processes cannot be excluded.

Furthermore, precise anamnestic data regarding the exact time since last watering, specific transport durations, and varying nutritional histories were unavailable. We mitigated this by utilizing serum osmolality and hematocrit as downstream, physiological integrators of the animal’s actual hydration state at the time of sampling. Additionally, we did not quantify endocrine stress markers (e.g., serum cortisol). Therefore, while our data captures the structural consequences of hemoconcentration, we cannot fully disentangle the specific morphological impacts of acute HPA-axis activation (e.g., transport stress) from chronic environmental dehydration. Finally, while official veterinary inspections confidently ruled out clinical disease and heavy parasitic burdens, the presence of minor subclinical infections cannot be absolutely excluded without comprehensive serological profiling. Future longitudinal studies incorporating endocrine panels are required to confirm these pathways. Additionally, future histomorphometric investigations should explicitly employ microvascular staining and sinusoidal quantification to formally test the hemodynamic vasoconstriction hypothesis proposed herein.

Second, while we differentiated chronic dehydration using body condition scores and regional analysis, we cannot fully isolate the acute stress of pre-slaughter transport. However, the significant morphological differences observed between the “Arid” and “Semi-Arid” populations, who underwent similar transport conditions, suggests that the observed remodelling is likely a result of chronic environmental pressure rather than acute handling alone.

Future studies should employ longitudinal or controlled dehydration–rehydration designs incorporating endocrine, metabolic, nutritional, inflammatory, and microvascular measurements to determine whether MZ remodeling is reversible, whether it is specific to hydric stress, and whether it has measurable consequences for splenic immune function.

Conclusion

The present study identifies the splenic MZ as a highly plastic, hydration-sensitive compartment in Camelus dromedarius. Severe dehydration was associated with marked MZ thinning, reduced MZ B-cell density, and coordinated changes in systemic hemoconcentration. The mediation model further indicated substantial statistical coupling between MZ thickness and B-cell density. However, the cross-sectional design does not establish that MZ atrophy causes B-cell depletion, confers a survival benefit, or represents a dehydration-specific adaptive mechanism. The observed remodeling may instead reflect the integrated effects of hydric, hemodynamic, metabolic, endocrine, nutritional, and other physiological stresses. These findings therefore provide quantitative evidence for dehydration-associated splenic plasticity and establish a foundation for future longitudinal and mechanistic studies of its reversibility, biological mechanism, and functional significance.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

MAF: Conceptualization, Data curation, Methodology, Software, Resources, Supervision, Visualization, Project administration, Writing  –original draft. AM: Conceptualization, Formal analysis, Resources, Visualization, Writing – original draft. NE: Conceptualization, Formal analysis, Investigation, Writing  –original draft. RM: Investigation, Project administration, Resources, Writing – original draft. AF: Writing  –review & editing. MC: Investigation, Writing  –original draft. BF: Formal analysis, Writing – original draft.

Funding

The authors declare that this study received no financial support or funding of any kind.

Data availability

Data will be made available on reasonable request.

Declarations

Ethics approval and consent to participate

Ethical review and approval were waived for this study because all samples were collected post-mortem from animals subjected to routine commercial slaughter for human consumption at state-approved municipal abattoirs. No animals were euthanized specifically for the purposes of this research. All ante-mortem handling and slaughter procedures were conducted under the strict supervision of official state veterinarians and in full compliance with the national animal welfare regulations and sanitary slaughter guidelines established by the Algerian Ministry of Agriculture and Rural Development.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.