Leishmaniases are among the six major worldwide diseases according to the World Health Organization (2022). In Brazil, cutaneous leishmaniasis occurs throughout the national territory. The great diversity of species characteristic of the Amazon Rainforest also extends to vectors, reservoirs, and Leishmania species, resulting in a complex eco-epidemiological scenario with overlapping niches, thus complicating the understanding of the parasite’s biology (Nolder et al., 2007). In the context of leishmaniasis in the Brazilian Amazon, Santarém municipality, Pará State, is particularly prominent due to the occurrence of both cutaneous and visceral forms, as well as the record of five out of the seven species causing cutaneous leishmaniasis in Brazil, and the presence of subpopulations of Leishmania (Viannia) shawi and seven hybrid lineages between L. (V.) guyanensis and L. (V.) shawi (Jennings et al., 2014; Lima et al., 2021).

Reproduction and population structure of organisms in the genus Leishmania is predominantly clonal (Tibayrenc, 1995; Tibayrenc and Ayala, 2012; Tomasini et al., 2014). With the advent of increasingly sophisticated molecular tools, knowledge regarding the reproductive mechanisms and the generation of diversity in the genus Leishmania has been continually evolving (Tibayrenc et al., 1991; Akopyants et al., 2009; Sterkers et al., 2011; Rogers et al., 2014).

Although sexual reproduction in the genus Leishmania is well documented, the frequency and contribution of sexual events to population dynamics are still widely debated (Bastien et al., 1992; Akopyants et al., 2009; Rougeron et al., 2009; Tibayrenc and Ayala, 2013). Moreover, little is known about how these events contribute to the phenotypic properties of hybrids, including the expression of factors that influence infectivity, virulence, and transmissibility, for example (Torrico et al., 1999). In vivo infection experiments, for example, have shown that hybrids can display enhanced infectivity and higher parasite burdens (Kelly et al., 1991; Delgado et al., 1997; Nolder et al., 2007; Romano et al., 2014). Hybrid parasites have also been reported to gain advantages in colonization and development within insect vectors, thereby increasing their transmission potential (Volf et al., 2007).

Given the limited availability of isolated hybrid parasites for studies investigating the impacts of natural genetic recombination between different Leishmania species on parasite biology and disease manifestation, this study aimed to characterize the infection profile induced by a Leishmania (Viannia) guyanensis/Leishmania (Viannia) shawi hybrid in susceptible mouse strains and to compare it with the profiles produced by each parental species.

The aim of this study was to evaluate the infection behavior of the hybrid L. (V.) guyanensis/L. (V.) shawi (M19672) in relation to the parental species. Infection by the hybrid parasite was found to occur earlier and progress faster compared to the parental strains, which exhibited slower lesion development, peaking at later time points. The evolution of the swelling was quite heterogeneous when comparing the three groups of parasites, with L. (V.) shawi causing significantly less swelling than the other two strains. Notably, L. (V.) guyanensis produced lesions with a diameter more than double that of the hybrid parasite. These findings suggest that the inheritance of virulence factors from both L. (V.) guyanensis and L. (V.) shawi led to lesions displaying an intermediate profile compared to those observed in the parental species. These results show that a single recombination event—even between closely related parental species—can induce biological changes, underscoring the need for broader investigations of hybrid lineages within the Leishmania genus.

For the cytokine analysis, the 35^th day post-infection was chosen due to the difference in lesion profiles among the three groups ( Figure 1 green arrow), as well as the prognosis for disease progression or regression. Thus, pro-inflammatory cytokines IFN-γ and IL-12, as well as anti-inflammatory cytokines IL-4 and IL-10, were quantified in the skin and lymph nodes of BALB/c mice. Infection with the hybrid parasite resulted in higher IL-4 production in the skin compared to the control group. IL-4 is classically associated with the Th2-type cellular immune response, which is linked to disease progression in the classical L. (L.) major infection model (Hurdayal and Brombacher, 2014). However, no progressive lesion was observed; instead, it was suggestively in the process of healing. The role of IL-4 has been questioned following evidence that it stimulates dendritic cells from BALB/c mice to produce IL-12 in the presence of Leishmania (Leishmania) major during the early stages of infection (Biedermann et al., 2001). Other studies suggest that IL-4 can be expressed in contexts of Leishmania infection even in the absence of clinical lesions, both in animal models and in humans (Baratta-Masini et al., 2007; Felizardo et al., 2012). Notably, at 35 days post-infection, L. (V.) guyanensis elicited higher IL-10 production in the skin than did the hybrid parasite. The difference in IL-10 production may reflect the progressive stage of the swelling caused by L. (V.) guyanensis and the regressive stage of the swelling observed in the paws of mice infected with the hybrid parasite. When examining swelling progression in the skin of the groups infected with L. (V.) guyanensis and L. (V.) shawi, higher IL-4 production supports the findings, as it was quantified in greater amounts in the first group, which exhibited clear disease progression, while the second group showed more discrete progression of skin impairment. Additionally, the production of IFN-γ in the lymph nodes of mice infected with the hybrid parasite was lower than that observed in the uninfected control group. This finding was similar to what was observed in the infection with the parental species L. (V.) guyanensis. The high concentration of IL-4 associated with low levels of IFN-γ observed in the group infected with the hybrid parasite and L. (V.) guyanensis has been described in other studies; however, it is classically associated with a progressive infection scenario (Szabo et al., 1997; Borges et al., 2018). Intriguingly, the group infected with the hybrid parasite displayed a profile indicative of resolution at the site of the swelling while maintaining parasitism similar to that of the other groups until late stages of infection.

In infection with L. (V.) guyanensis, higher production of the cytokine IL-4 was observed both in the skin and in the draining lymph nodes compared to the uninfected control group. The promotion of a Th2 response by IL-4 has been associated with macrophage activation toward the polyamine and arginase pathways, favoring parasite persistence, as well as with the inhibition of IL-12 production, which would otherwise recruit Th1 cells to the infection site (Kropf et al., 2005). The data obtained from the experimental infection of BALB/c mice with L. (V.) guyanensis indicates a Th2-type response profile, with a predominance of IL-4 and the presence of IL-10, which apparently favored swelling progression during the chronic phase of the disease (Himmelrich et al., 1998). Th1-type cytokines did not show significant differences compared to uninfected mice in the skin. However, in the lymph nodes, the L. (V.) guyanensis-infected group showed lower IFN-γ production compared to the uninfected group. Although still underexplored, the infection profile of L. (V.) guyanensis, both in experimental models and human infections, has been described as more related to susceptibility (Martinez et al., 1991; Matta et al., 2009; Gomez et al., 2018). Infection experiments in susceptible murine models have shown that lesion progression in BALB/c mice peaks around the 5th week post-infection (Matta et al., 2009), which supports our findings.

During experimental infection with L. (V.) shawi, swelling progression remained limited until day 28 post-infection. By 35 days post-infection, slight paw swelling was observed; however, it did not progress to a typical lesion as seen with other species. Despite the characteristic infection control profile, there was a similar parasitic load to the other groups and no predominance of Th1-type cytokines in the skin of the mice.

The substantially higher parasite load values observed for the hybrid strain at the upper time limit of the experiment allowed for the selection of a linear model to describe its temporal dynamics. However, the trend curves—even based on log-transformed data—indicate that both parental strains exhibit a reduction in parasite load at some point between days 38 and 60 (corresponding to the peak and the upper time limit of the experiment, respectively). Integrating the statistical analysis results, a similar temporal pattern in parasite load dynamics could be observed between L. (V.) shawi and L. (V.) guyanensis, with a peak at day 38 followed by a decline toward day 60. In contrast, the hybrid strain exhibited a significantly higher parasite load compared to both parental strains and followed a distinct temporal pattern, with a continuous and significant increase up to day 60. These findings may indicate an adaptive advantage of the hybrids related to increased transmissibility. It is noteworthy that these parasites were isolated from patients with ATL in Santarém (PA), where they are part of the disease transmission cycle (Jennings et al., 2014).

A possible hypothesis is that L. (V.) shawi may remain silent in BALB/c mice due to the absence of virulence factors or possibly due to a tolerance induction pathway. This hypothesis could also account for the persistent parasitemia observed in mice infected with the hybrid parasite, despite the Th2-skewed cytokine profile in their skin. In the experimental infection model of C57BL/6 mice with L. (L.) major, parasite persistence in the host is associated with a population of regulatory T cells through IL-10-dependent or independent mechanisms (Belkaid et al., 2002). Further studies analyzing cytokines such as IL-5, IL-13, and regulatory T cell populations are needed to clarify the data obtained from BALB/c mice infected with L. (V.) shawi. Another important approach of investigation in this context would be the analysis of genes associated with virulence factors and their expression profiles among the parental species and the hybrid isolate M19672. It is important to note that experimental infection data for the parental species in BALB/c mice are scarce; consequently, our findings advance understanding of their behavior and provide a foundation for future studies.

In conclusion, the experimental infection model using L. (V.) guyanensis, L. (V.) shawi, and their hybrid in BALB/c mice revealed distinct infection profiles, with the hybrid lineage exhibiting intermediate virulence profile, and high parasite loads even in the absence of lesions.

Our findings highlight that a single genetic recombination event can substantially alter parasite biology, potentially affecting virulence and immune modulation. These results provide new insights into the complexity of host-parasite interactions in Leishmania infections and emphasize the need for further studies focusing on the role of hybridization in disease pathogenesis and parasite persistence.