Objective: We contribute to the understanding of the transmission dynamics of Leishmania infantum suggesting the involvement of rabbits as wild reservoirs. Results: The prevalence of infection was 86.0% (270/314 wild rabbits) ranging from 18.2% to 100% in natural geographical regions. The estimated average parasite load was 324.8 [CI 95% 95.3-554.3] parasites per mg of ear lobe ranging from 0 to 91,597 parasites/mg per tissue section. Conclusions: A positive correlation was found between skin parasite load in wild rabbits and human incidence with evidence of the presence of the same L. infantum genotypes in rabbits and humans, providing new epidemiological and biological basis for the consideration of wild rabbits as a relevant L. infantum wild reservoir. Molecular parasite surveillance reflects the great genotypic variability of the parasite population in wild rabbits. Most of these genotypes have also been found to infect humans, dogs and sandflies in the region. Our findings also highlight that direct genotyping of the parasite in host tissues should be used for molecular surveillance of the parasite instead of cultured isolates.

Background: Vector-borne diseases such as leishmaniasis exert a huge burden of morbidity and mortality that are mainly controlled through vector control. The increasing threat of insecticide-resistant vectors entails incorporating more vector control interventions to eliminate these diseases. Introduction of Wolbachia into wild vector populations has been suggested as a potential vector control measure that would require extensive regional knowledge. The aim of this work is to estimate the prevalence of Wolbachia infection and monitor circulating strains in wild sand fly populations from Spain and Morocco, two countries where leishmaniasis is endemic. Methods: Wolbachia was detected using polymerase chain reaction (PCR). Haplotype diversity was performed by sequencing, and phylogenetic relationships were then established. In silico prediction of the Wolbachia surface protein (WSP) structures was performed. To investigate the relationship between epidemiological variables and the presence of Wolbachia, regression analyses were employed. Results: Wolbachia was detected in 45.8% of the specimens tested (319/697), and similar infection rates were found (P = 0.92) in males (46.1%; 94/204) and females (45.6%; 225/493). Differences in infection were detected among Spanish sand fly species (P < 0.001), being higher for Phlebotomus papatasi (35/52) and Phlebotomus perniciosus (239/384). No infected Phlebotomus sergenti specimens were found in Spain, whereas two different Wolbachia haplotypes were detected in P. sergenti sand flies from Morocco. No significant differences were found between sex, species, or capture sites in specimens captured in Morocco (P > 0.05). Five Wolbachia haplotypes distributed in the known A and B supergroups were identified. Structural analysis showed a nine-amino acid insertion in the fourth loop of a Wolbachia haplotype found in P. sergenti specimens from El Borouj (Morocco). Conclusions: We confirmed the circulation of different Wolbachia strains in all sand fly species investigated. All L. infantum proven or suspected vectors shared the same, or a closely related, Wolbachia haplotype. The haplotype bearing the loop insertion was found in the locality undergoing an anthroponotic cutaneous leishmaniasis outbreak. These extracellular loops might have some role in enhancing or inhibiting the development of Leishmania and other pathogens in sand flies. These findings are very promising and highlight the need to further investigate the tripartite interactions between Wolbachia strain, Leishmania species, and sand fly species/lineage. Graphical

Leishmaniasis by Leishmania infantum has a zoonotic transmission cycle involving an increasing number of mammalian hosts, forming a cooperative network. The sand fly feeding on livestock is evidenced, but clinical confirmation regarding their infection is limited. We aimed to evaluate Leishmania seroprevalence in livestock to assess its impact on leishmaniasis epidemiology in an endemic area located in the Mediterranean region. A cross-sectional serological study screened livestock exposure to L. infantum and risk factors in Southern Spain. A total of 864 serum samples of clinically healthy sheep, goats, cattle, and pigs were examined by an indirect fluorescence antibody test, using a 1/80 cut-off titre to minimize cross-reactions. Global seroprevalence was 10.8%: 21.6% cattle, 15.4% sheep, 7.3% goats, and 0.6% pigs. Statistically significant differences in positive detection were observed among species (p < 0.001) and natural regions (p < 0.001). High positive reactions in cattle, goats, and sheep suggest livestock exposure to Leishmania spp. and potential asymptomatic infection. Livestock presence in biotopes could promote a dilution effect, reducing human leishmaniasis incidence. Further investigation is needed to confirm livestock roles in leishmaniasis maintenance and transmission.

Background: Phlebotomine sand-fly-borne infections are an emerging threat to human and animal health in Mediterranean countries, highlighting the need for improved surveillance and control strategies. Dogs are ideal sentinel hosts owing to their central role in the transmission of zoonotic Leishmania infantum , frequent exposure to sand fly bites, capacity to develop antibodies to phleboviruses, and close contact with humans. This study reports cross-sectional surveys of antibodies to Leishmania , Toscana virus (TOSV), and Sicilian sand fly virus (SFSV) in dogs from Portugal, Spain, Italy, Croatian Istria, Turkey, and Israel, as well as antibodies to salivary proteins of Phlebotomus perniciosus (Portugal, Spain, and Italy) and Phlebotomus papatasi (Spain and Italy), conducted within the Climate Monitoring and Decision Support Framework for the Detection and Mitigation of Sand fly Diseases with Cost–Benefit and Climate Policy Measures (CLIMOS) project. Methods: Blood samples and epidemiological data were collected from 2500 dogs. Antibodies to Leishmania were detected by indirect immunofluorescence, phlebovirus antibodies by seroneutralization assays, and antibodies to P. perniciosus and P. papatasi salivary antigens by enzyme-linked immunosorbent assay (ELISA) using recombinant proteins rSP03B and rSP36, respectively. Sources of antibody variability were evaluated using mixed-effects logistic regression models. Results: Antibodies to L. infantum , phleboviruses, and sand fly saliva were widely detected, although seroprevalence varied markedly by region. No Leishmania -seropositive dogs were found in Istria, parts of northern Spain, or several districts in Israel, whereas seroprevalence exceeded 30% in Sicily and in several Turkish and Spanish provinces. TOSV seropositivity was generally absent or below 5%, except in southern Spain (8–24%) and Muğla, Turkey (10%). SFSV exposure was highly focal, occurring mainly in Turkey (12%), Israel (12%), and Lisbon (7%). Exposure to P. perniciosus was very high in Portugal, Sicily, and most of Spain, while P. papatasi exposure was highest in Sicily and selected Spanish regions. Antibody variability was driven primarily by geographical location. Conclusions: The marked geographical heterogeneity observed confirms dogs as valuable sentinels for sand-fly-borne infections. These infections are highly clustered across Mediterranean regions, likely reflecting differences in sand fly density and infection rates. Understanding the drivers of this heterogeneity is essential for accurate risk mapping and effective control strategies.

Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania, which is endemic in certain areas of Europe, such as southern Spain. The disease manifests in various clinical phenotypes, including visceral, cutaneous, mucosal, or asymptomatic leishmaniasis. This diversity in clinical outcomes may be influenced by the host immune response, with human leukocyte antigen (HLA) molecules playing a crucial role in determining susceptibility and progression of the infection. This study explores the association between specific HLA variants and Leishmania infantum infection. We recruited four cohorts: a control group, asymptomatic individuals, patients with symptomatic disease, and cohabitants of infected individuals. HLA typing was performed for all participants, followed by an association analysis with infection status and disease progression. Our findings indicate that the HLA-B*38 and HLA-C*03 alleles are associated with protection against L. infantum infection. These results contribute to a better understanding of the disease’s progression, offer potential for new therapeutic approaches such as vaccines, and expand the existing knowledge in the literature.

The objectives of this study were to establish a spatial framework and monitor a Mediterranean leishmaniasis hotspot over time and identify clinical leishmaniasis risk factors through comparative analysis of patient habits, housing conditions, and entomological surveillance data. The study was conducted in southern Spain. The data set (from 2003 to 2024), was derived from various sources, including notification systems, hospital databases, and patient and family surveys. Sand flies were collected using CDC and sticky traps in and around the households of 8 cases. Patients and family members were surveyed using questionnaires that addressed demographic characteristics, housing conditions (including animal contact), and daily habits. A logistic regression analysis was performed to identify variables associated with clinical leishmaniasis. The incidence rates of human leishmaniasis were 15.1 (Montefrío) and 8.3 (Alcalá la Real). Phlebotomus perniciosus was the most abundant species (83.4%). Household infestation was detected in 100% of dwellings (both, indoors/semi-indoors, and patios, terraces, or gardens) and in 71.4% of nearby walls. The abundance index was 10.8, 24.6, and 14.0 P perniciosus/household, respectively. The Leishmania infantum infection rate in P. perniciosus was 24.0% (30/125). Final multivariate model showed that clinical leishmaniasis was associated with age, immunosuppression, number of adult residents, second residence, nighttime fan use, and indoor insecticide use. This study demonstrates the presence of a high-incidence hotspot and local transmission of Leishmania infantum in the households with treated cases. To our knowledge, this is the first study to assess risk factors associated with clinical leishmaniasis caused by L. infantum in southern Europe.