In tropical and subtropical areas, global health is greatly impacted by parasitic diseases caused by protozoan parasites [1] . Leishmaniasis and malaria are two of the most prevalent of them affecting millions yearly. Cutaneous leishmaniasis caused by Leishmania major is characterized by localized skin lesions, permanent scarring and ulcerations [2] [3] . Due to its immunological parallels to human illness, several studies on L. major have been conducted in mouse models [4] . Contrastingly, Plasmodium berghei (P. berghei) is a rodent malaria parasite that mimics P. falciparum. According to [5] , P. berghei has been a valuable model in investigating immune responses, novel antimalarial regimens and host-pathogen interactions.

Polyparasitism is rather common in endemic regions where people harbour several parasites. This has the power to drastically change the course of disease development, pathophysiology and treatment outcomes [6] . The majority of experimental models, however, examine these infections separately, perhaps ignoring intricate host-pathogen dynamics [7] [8] . Through the modulation of systemic immune responses, the erythrocyte-tropic P. berghei and skin-dwelling L. major may affect each other’s replication and pathogenicity. For instance, L. major triggers Th1 immunity while P. berghei triggers both Th1 and Th2 responses [9] .

To enhance diagnostic and treatment approaches, it is indispensable to understand how these disparate immune responses interrelate during co-infections. Specifically, little is known about the impact of co-infection on systemic measures that are essential for determining the severity of the disease and host tolerance, such as organ pathology. In addition to providing baseline data for creating integrated disease management strategies in co-endemic areas, this study will further our understanding of host responses to protozoan co-infections.

The current study investigated the impact of Leishmania major (a cutaneous leishmaniasis-causing protozoan), Plasmodium berghei (a rodent malaria parasite), and their co-infection on the pathology of mice.

The granuloma formation observed in the liver, spleen and brain tissues in the Leishmania major-infected group aligns with prior studies demonstrating hepatic granulomatous responses in visceral and cutaneous leishmaniasis, even in non-visceral strains, due to hepatic parasite migration or antigen deposition [11] - [13] . Similar studies using Leishmania donovani in mice and hamsters have documented granulomas composed of mononuclear cells, especially Kupffer cells, with concurrent hepatocyte damage and fibrosis [12] .

The liver pathology seen in P. berghei-infected mice is consistent with findings from malaria studies in rodent models. Hemozoin accumulation within Kupffer cells and sinusoids, hepatic congestion, and hepatocyte degeneration are hallmark features reported in both P. berghei and P. chabaudi infections [14] [15] . These changes are associated with systemic inflammation, hypoxia, and the hepatocellular stress response triggered by parasitized red blood cells and inflammatory cytokines.

Interestingly, the co-infection model showed histological features of both pathogens but with potentially moderated responses. For instance, while immune infiltration and hepatocyte damage were severe, granuloma formation and hemozoin deposition appeared less pronounced than in single infections. This suggests an immunological interaction where L. major infection may alter the hepatic microenvironment or modulate the host immune response to P. berghei, reducing pigment accumulation and fibrotic changes. Comparable immunomodulatory effects have been described in co-infection studies, such as Leishmania-Trypanosoma or Leishmania-Plasmodium models, where altered cytokine profiles and macrophage activation states impact pathology [3] [9] .

The observation that L. major co-infection appeared to reduce cerebral hemozoin deposition in P. berghei-infected mice suggests that the dermotropic parasite may alter host immune dynamics in a way that modifies malaria pathogenesis. One potential mechanism involves macrophage activation states. L. major infection drives strong macrophage responses that can skew toward either a classical (M1) or alternative (M2) phenotype depending on host genetics and cytokine milieu [16] [17] . An M1-skewed response, enriched in IFN-γ, TNF-α, and nitric oxide, could enhance phagocytic clearance and degradation of parasitized erythrocytes, thereby limiting hemozoin accumulation [9] . Conversely, M2-associated cytokines such as IL-4 and IL-10 might dampen the excessive inflammatory activation that typically fuels malaria-associated pathology, thus modifying hemozoin deposition indirectly [18] .

Similarly, cytokine cross-regulation between the two infections may contribute. Leishmania major infection is known to induce strong Th1-associated cytokines (IL-12, IFN-γ) in resistant strains or Th2-associated cytokines (IL-4, IL-10) in susceptible strains [19] [20] . In the context of P. berghei co-infection, a Th1-dominant profile could improve parasite control and erythrocyte clearance efficiency, while IL-10 production could restrain hyperinflammation and reduce monocyte-driven hemozoin accumulation in cerebral microvessels [21] . Furthermore, chemokines such as CCL2 and CXCL10, upregulated during leishmanial infection, may alter leukocyte trafficking into the brain and change the cellular niche where hemozoin is deposited.

In L. major-infected mice, the disruption of splenic architecture, particularly the blurring of the marginal zone and hypertrophy of lymphoid follicles, reflects an active humoral immune response. Such disorganization is consistent with prior observations in Leishmania donovani infections, where splenic white pulp disintegration and follicular atrophy are linked to chronic antigenic stimulation [16] . Though L. major is typically considered dermotropic, this study reinforces the systemic immunopathological impact it can exert during sustained infection.

The presence of megakaryocytes and fibrotic changes signifies extramedullary hematopoiesis and chronic inflammation, respectively. Similar findings [17] noted increased megakaryocyte activity in the spleens of mice infected with Leishmania, possibly reflecting a host response to anemia or bone marrow suppression. The observed collagen deposition and fibroblast infiltration align with studies showing that chronic leishmaniasis induces stromal remodeling in lymphoid organs [9] [18] .

Interestingly, the detection of hemozoin pigment in L. major-infected spleens, albeit moderate, may suggest macrophage activation or prior immune priming. While hemozoin is typically a byproduct of hemoglobin digestion by malaria parasites, reports such as [19] [20] indicate that macrophages in leishmaniasis can sequester pigment remnants from previous co-infections or systemic inflammation. The splenic pathology in P. berghei-infected mice is characterized by white pulp hypertrophy, germinal center enlargement, and significant red pulp infiltration. These features are hallmark responses to malaria-induced hyperactivation of the immune system. [20] reported similar germinal center changes in murine models of cerebral malaria, attributing them to robust B-cell responses and cytokine storms.

Prominent hemozoin accumulation, a well-established malaria pathology marker, was widely distributed in the splenic tissue in our study. This finding is in line with prior studies [21] [22] that describe hemozoin as both a diagnostic marker and an immunomodulatory agent contributing to systemic inflammation. The fibrotic changes and sinusoidal dilation observed here suggest tissue adaptation to prolonged immune activation and vascular stress, confirming findings [23] on chronic splenic damage in Plasmodium-infected rodents. Megakaryocyte infiltration again supports the presence of reactive extramedullary hematopoiesis a phenomenon documented in severe and chronic malaria as a response to anemia and bone marrow suppression [15] .

The co-infected group exhibited the most profound architectural disruption and immune dysregulation, with total obliteration of red/white pulp boundaries, excessive lymphoid follicle expansion, and intense immune cell infiltration. These results illustrate an additive or synergistic pathological impact, consistent with the hypothesis that polyparasitic infections exacerbate host tissue damage. The combined presence of degenerative features (e.g., vacuolation, cell lysis), high-density immune clusters, abundant megakaryocytes, and fibrotic remodeling is suggestive of overwhelming immune stimulation and chronic inflammation. Studies support the view that co-infections can dysregulate immune homeostasis, leading to both overactivation and exhaustion [3] [24] .

Furthermore, the enhanced hemozoin burden and fibroblast infiltration indicate both active malaria pathology and long-standing tissue damage and repair processes, respectively. These findings underscore the complex interplay between immune responses to different parasites and the resulting structural compromises in lymphoid organs. The mild disruption in brain architecture observed in L. major-infected mice, including fibrous deposition, immune infiltration, and granuloma formation, aligns with earlier reports indicating that while L. major is primarily dermotropic, it can induce neuroinflammatory changes under certain conditions. [25] reported microglial activation and glial proliferation in the cerebral cortex of Leishmania donovani-infected rodents, which is consistent with the activated microglia and gliosis noted in our study. However, our data suggest that even in cutaneous leishmaniasis, such as with L. major, systemic inflammatory responses may extend into the Central Nervous System (CNS), albeit to a milder degree compared to visceralizing species.

The mild disruption of brain architecture in Leishmania major infected mice characterized by fibrous deposition, immune cell infiltration, granuloma-like foci, and accompanying microglial activation with gliosis supports the emerging view that Cutaneous Leishmaniasis (CL), despite its predominantly dermotropic tropism, can exert measurable effects on the Central Nervous System (CNS) under specific inflammatory milieus. Prior studies in Visceral Leishmaniasis (VL), especially with L. donovani, have documented microglial activation, glial proliferation, and perivascular inflammatory changes in rodent models; your findings are concordant with this neuroinflammatory signature but scaled down in magnitude, consistent with the lower systemic parasite burden and tissue dissemination typical of L. major relative to visceralizing species.

Furthermore, the neuronal alterations observed, such as pyknosis, vacuolation, and nuclear lysis are similar to those seen in chronic neuroinflammatory conditions and suggest potential neurotoxic effects mediated by cytokines or parasite-derived molecules. This aligns with [26] , which demonstrated that cytokine storms induced by chronic Leishmania infection can cause neural stress and cellular damage in peripheral and central nervous tissues. Our findings in P. berghei-infected mice revealed hallmark features of cerebral malaria, including widespread BBB breakdown, perivascular leukocyte infiltration, and deposition of hemozoin. These results are in agreement with previously documented cerebral pathology in murine models of cerebral malaria (SCM). [27] [28] described severe vascular pathology and widespread neurodegeneration in P. berghei ANKA-infected C57BL/6 mice, with extensive microglial activation and parasite sequestration in brain capillaries findings that mirrored our observations of microglial clustering and hemozoin-laden vessels.

The co-infected mice demonstrated exacerbated neuropathology, surpassing the severity seen in single infections. This synergistic effect supports the hypothesis that co-infections can exacerbate inflammatory and neurodegenerative processes. Previous studies examining co-infection effects have shown that concurrent parasitic infections can heighten systemic immune activation, leading to more pronounced tissue damage [29] [30] . Notably, the compounded effects on BBB integrity, immune infiltration, and neuronal loss in our study suggest that co-infection intensifies CNS pathology via both immune-mediated mechanisms and direct parasitic insults. Hemozoin deposits, extensive microgliosis, and severe glial proliferation in co-infected mice provide compelling evidence of an additive or possibly synergistic neuropathogenic interaction between Leishmania and Plasmodium.

Co-infection with Leishmania major and Plasmodium berghei caused more severe tissue damage than either infection alone. In the liver, mice showed extensive granuloma proliferation, immune cell infiltration, hepatocyte degeneration, and vascular congestion. Spleens showed disruption of red/white pulp boundaries, leukocytic infiltration, and fibrosis. Brain sections showed severe blood-brain barrier disruption, hemorrhage, and neuronal degeneration. Future studies should investigate immunological interactions driving synergistic tissue damage. Histological descriptions of fibrous deposition, microglial activation, and granuloma formation provide qualitative evidence, but without detailed quantitative morphometry, immunohistochemistry, or imaging, subtle differences may be under- or over-estimated.

We would like to acknowledge Dr. Benjamin Mwongela, Mr. Kenneth Waititu, Mr. Thomas Adino, Mrs. Priscilla Kimiti, Ms. Gladys Korir and Mr. Dominic Mwaura from the Animal Science Department, KIPRE, without whom this study would not have been a success.

Approval for the project was sought from the Institutional Scientific and Ethical Review Committee (ISERC) of the Kenya Institute of Primate Research (KIPRE) (study ISERC/10/2016). Animal experiments were recognized under great care and the protocols were conformed under the Institutional Animal Care and Use Committee (IACUC) and included the internationally accepted standards of conducting animal researches. There were humane endpoints in the course of the study to reduce suffering in animals.