Retrospective entomological and eco-epidemiological survey of a leishmaniasis outbreak in southern Spain

Abstract

Leishmaniasis is a well-known vector borne disease with a high incidence in southern European countries. In Spain, Leishmania infantum infections typically occur as sporadic and infrequent outbreaks. However, in recent years, several outbreaks have been reported in different regions, accompanied by increasing hospitalization rates. In 2023, 31 human cases of Leishmania infection were reported in hospitals across 15 municipalities in Córdoba (southern Spain). Visceral leishmaniasis was the predominant form (77.4%), followed by cutaneous (19.4%), and mucocutaneous (3.2%) forms. A retrospective analysis of Phlebotomus captures from the mosquito monitoring network was conducted to characterize the phenology, abundance, and feeding behaviour of sand flies in the main outbreak area. A total of 22,220 sand fly specimens were recorded, with male identification revealing five species: Phlebotomus perniciosus (94.3%), Sergentomyia minuta (4.3%), Phlebotomus ariasi (0.08%), Phlebotomus sergenti (0.03%), Phlebotomus papatasi (0.005%) and 1.3% undetermined. Sand fly captures peaked in September, preceding the major peak in human case notifications. Most of the identified blood meals (75.0%, n = 163) came from Iberian rabbits (Oryctolagus cuniculus algirus), although blood from six other mammal species, including two samples of human origin, and one bird species was also detected. A total of 5855 female sand flies analysed, grouped into 145 pools, showed a Leishmania infection prevalence of 3.8%, peaking in October and November. Our findings suggest that the Iberian rabbit may play a key role in the local epidemiology of L.infantum, given its high frequency as a blood source for Ph. perniciosus and the elevated vector densities and infection rates observed in the outbreak area. These results emphasize the need for continuous entomological surveillance and proactive vector management, integrating the monitoring and control of potential reservoir hosts to effectively reduce transmission risk in the affected areas.

Highlights

• •Leishmania infantum outbreak is reported in southern Spain in 2023.
• •Phlebotomus perniciosus was the predominant sand fly species (94.3%).
• •European rabbits were the main blood source for Phlebotomus perniciosus.
• •Leishmania infection in sand flies peaked during October–November.

1. Introduction

In the Mediterranean basin, the zoonotic form of leishmaniasis is predominantly caused by Leishmania infantum [1], which circulates between animal reservoirs and sand fly vectors, maintaining stable endemic transmission [[2], [3], [4]]. In Spain, cutaneous, mucocutaneous, and visceral forms of human leishmaniasis caused by L. infantum are present, with dogs serving as the main reservoir [5,6]. Recently other wild mammals, primarily lagomorphs, have also been found infected with L. infantum [7]. Leishmaniasis transmission is primarily attributed to two sand fly species in the country, Phlebotomus perniciosus and Phlebotomus ariasi.

The epidemiology of leishmaniasis in Spain has shifted notably over the past two decades [8]. The largest outbreaks of human leishmaniasis in Europe occurred in 2010 in the southwest of Madrid, since then, nearly 850 cases have been reported, with incidence rates spiking at up to five times the average of the previous years [[9], [10], [11]]. The average annual hospitalization rate for leishmaniasis increased from 0.4 to 0.6 cases per 100,000 inhabitants between 1999 and 2003 [12] to 0.8 by 2023 [13]. A recent analysis of Leishmania incidence during 2016–2017 estimated rates of 0.79–0.86 per 100.000 inhabitants for visceral form, 0.88–1.04 for cutaneous form, and 0.12 for mucocutaneous form [2].

In 2023, Andalusian health authorities reported multiple leishmaniasis human cases in both rural and urban foci throughout the province of Córdoba (south Spain). The area where human cases were reported geographically overlapped with four trapping sites that were part of the West Nile virus mosquito surveillance network operated by the Consejería de Salud y Consumo - Junta de Andalucía (Andalusian Regional Health Authority). Given that sand flies are also attracted to the mosquito traps used, a retrospective study was conducted on Leishmania vector captures at these sites to characterize the entomological and epidemiological factors involved. This included morphological and molecular analysis of sand fly samples collected in the affected area, as well as characterization of sand fly feeding networks during the leishmaniasis outbreak.

2. Material and methods

2.1. Human leishmaniasis notification

In Spain, all forms of human leishmaniasis have been notifiable diseases since 2014, and clinicians are required to report confirmed or suspected cases to public health authorities. Data on human leishmaniasis cases were obtained from the Consejeria de Salud y Consumo, which compiles notifiable diseases reported by healthcare professionals across Andalucía, a region with around 8,6 million inhabitants in south Spain. Only cases of patients reporting symptoms within the 2023 calendar year in municipalities of the province of Córdoba (Fig. 1) were considered in the analysis. Leishmania species identification was performed in hospital laboratories using standard molecular diagnostic techniques, including PCR-based assays targeting species-specific DNA regions, for 24 of the 31 human cases.

Fig. 1.

Geographic distribution of human leishmaniasis cases and sand fly sampling sites in Córdoba province (Andalusia, southern Spain) during 2023. Municipalities are shaded according to the number of human leishmaniasis cases reported (darker tones indicate higher case numbers). Black dots represent the four sand fly sampling locations (from top to bottom): Córdoba city, Fernán Núñez, Montalbán de Córdoba, and Aguilar de la Frontera.

2.2. Retrospective entomological study

Sand fly trapping was conducted in four locations of Córdoba province: Aguilar de la Frontera (37,533,834, − 465,270), Montalban de Córdoba (37,589,535, − 4,752,048), Fernán Nuñez (37,691,499, − 4,745,191), and Córdoba (37,843,768, − 4,809,138). These sites are situated within the region affected by the 2023 leishmaniasis outbreak (Fig. 1) and are predominantly rural areas surrounded by agricultural landscapes adjacent to small urban centres. Córdoba province, located in southern Spain, is characterized by a Mediterranean climate, with hot, dry summers and mild, wet winters, and pronounced seasonal variability in temperature and precipitation.

Since 2022, the Consejería de Salud y Consumo has operated a mosquito surveillance program comprising six monitoring stations across Córdoba province. Although this network was primarily designed for mosquito monitoring, the traps also captured other hematophagous dipterans, including sand flies. Sampling was carried out every two weeks using BG-Sentinel traps (Biogents, Germany) baited with approx. 1 kg of CO₂ (dry ice, Air Liquide, Spain) from the 3rd week of June 2023 to the 1st week of November 2023. All captured specimens were stored dry at −80 °C until further processing. Following the detection of a high number of human leishmaniasis cases, sand fly specimens from four stations overlapping the affected area were retrospectively analysed to assess their abundance, species composition, feeding behaviour, and Leishmania infection prevalence.

Sand flies were sorted on a Petri dish placed on an ice-cooled tray under a stereomicroscope. Due to the exceptionally high number of specimens, among the largest sand fly captures ever reported in Europe, a counting estimation method was applied to ensure efficiency and consistency. When more than 500 specimens were present, sand flies were evenly distributed across the Petri dish, and males and females present in a quarter of the total surface area were counted and extrapolated to the entire surface. The deviation between extrapolated and full counts in 10 plates was below 5%, confirming the robustness and reliability of this approach.

Blood-fed females were separated from non-blood-engorged ones. The latter were sorted by genus (Sergentomyia or Phlebotomus) [14] and pooled in groups of up to 50 females of the same genus, collected at the same site and date. Thus, pools contained only specimens of a single genus and were never mixed between Phlebotomus and Sergentomyia. The genitalia of all males (n = 2450) were mounted on permanent microscope slides using Hoyer's medium and examined under a compound microscope (Zeiss, Axioscope, UK) for species identification using stablished keys [15,16]. Undetermined specimens corresponded to damaged individuals lacking diagnostic morphological structures and therefore could not be reliably identified to species level.

2.3. Molecular screening of Leishmania DNA

A total of 145 pools, comprising 5855 female sand flies, were analysed for Leishmania detection. Pools were selected to evenly represent all sites and sampling dates. Each pool was homogenized in sterile DMEM buffer supplemented with 10% of fetal bovine serum (FBS), 0.5% of penicillin, and streptomycin, and 10% l-glutamine (Sigma-Aldrich, USA). Nucleic acids were extracted using QIAamp Viral RNA Mini Kit (QIAGEN, USA) [17], originally intended for ongoing phlebovirus studies; however, these nucleic DNA-rich extracts were also used for Leishmania detection. The presence of Leishmania in the sand fly pools was tested by amplifying a conserved 120 bp region of the kinetoplast minicircle DNA (kDNA) using primers JW11 (forward: 5′- CCTATTTTACACCAACCCCCAGT-3′) and JW12 (reverse) (5′- GGGTAGGGGCGTTCTGCGAAA-3′) [18]. Briefly, each PCR reaction consisted of 2 μL of 1× buffer, 0.8 μL of MgCl₂ (2 mM), 0.2 μL of 100 U/mL dNTPs, and 0.5 μL of HotSplit polymerase (final concentration 2.5 U/mL), 1 μL of JW11 and 1 μL of JW12 (final concentration 0.5 pmol each), 0.8 μL of BSA (final concentration 0.8 mg/mL; Roche®, Switzerland), and a variable volume of DNA adjusted to 30 ng according to the concentration values of the sample obtained by the NanoDrop™ (Wilmington, USA). DNase-free water was then added to make up a final volume of 20 μL. Positive controls consist of 1 μL of DNA (10 pg DNA/μL) of L. infantum, and each PCR batch included a negative control.

To characterize the Leishmania species, a fragment (∼350 bp) of the internal transcribed spacer 1 (ITS1) of ribosomal RNA was amplified using primers MEST1 (forward: 5′-CTG GAT CAT TTT CCG ATG-3′) and MEST2 (reverse: 5′-TGA TAC CAC TTA TCG CAC TT-3′) [19]. PCR was performed following the protocols described by El Tai et al. [19], with minor modifications to adapt it to the reagents used in this study: 1× Buffer, 1.5 mM MgCl2, 100 μM dNTPs mixture, 5 U of Hot split polymerase (Biotools B&M Labs, Spain), 1 μL of BSA DNase Free (20 mg/ml, Roche, Basel, Switzerland) and 25 pmol of each primer. PCR conditions were: one cycle of 6 min at 94 °C, 32 cycles consisting of denaturation at 95 °C for 20 s, annealing at 53 °C for 30 s and elongation at 72 °C for 1 min; final elongation was at 72 °C for 10 min. Negative and positive controls were included in each run using L. infantum. Half of the PCR products were resolved by electrophoresis in 1.5% agarose gels stained with CONDASAFE (CONDA) and visualized under UV light. In the case of ITS1 bands obtained in gels, the rest of PCR products were purified with illustra™ ExoProStar™ 1-Step (GE Healthcare Life Sciences).

2.4. Molecular identification of blood-meals and sand fly species

Blood-fed females (n = 338) were separated individually and classified on a scale based on both the volume (I – VI) and coloration of the blood meal (red, mixed, or dark) (Fig. 2). Each specimen was manually crushed with sterile lancets, and genomic DNA was extracted using the Maxwell® automated extraction system and the 16Lev Blood DNA kit (Promega, Madison, USA). Host identification was performed by the amplification and sequencing of a fragment of 748 bp of the cytochrome c Oxidase Subunit I (COI) mitochondrial gene [20].

Fig. 2.

Scale used to score the degree of blood engorgement in female sand flies from hardly visible engorgement (I) to full engorgement (VI), with progressive redness and abdomen distension reflecting the volume of blood intake. On the right, the three different colour categories (R = fresh red, M = mixed, and B = black). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

To confirm the identity of sand fly species associated with blood meals, a 658 bp fragment of the mitochondrial COI gene was amplified using universal primers LCO1490 and HCO2198 following Folmer et al. [21]. Sequences showing ≥98.5% homology with reference sequences in GenBank were considered valid for species identification.

2.5. Bioinformatics and statistical analysis

Amplicons were sequenced bidirectionally via capillary electrophoresis by the Genomics Service at Universidad Complutense de Madrid using an ABI PRISM 3730XL DNA Analyzer (Applied Biosystems, EEUU). The resulting electropherograms were manually inspected and corrected using ChromasPro program and Geneious v2020.0. [22] to generate consensus sequences. These sequences were then compared against the NCBI GenBank database using the BLASTn algorithm (http://www.ncbi.nlm.nih.gov/BLAST) to identify nucleotide fragment homologies. Finally, to identify the obtained nucleotide fragments, homologies with the available sequences data in GenBank were carried out with the software BLAST (http://www.ncbi.nlm.nih.gov/BLAST).

Data analyses were conducted in R (version 4.4.2). The prevalence of Leishmania infection in sand flies was estimated using the Maximum Likelihood Estimate (MLE) method [23] implemented in the PoolPrev package (version 0.1.3), which calculates infection prevalence and provides corresponding credible intervals. To assesses the relationships between locality, month, and infection status (presence/absence), we fitted a generalized linear model (GLM) with a binomial error distribution and a logit link using the base stats package. Model fit was evaluated through goodness-of-fit tests and residual diagnostics. Samples collected in October and November were excluded from these analyses because all pools from those months tested positive, but the sample size was too low resulting in extremely wide credible intervals and unreliable estimates. All visualizations were generated with ggplot2 package.

3. Results

3.1. Human leishmaniasis cases

A total of 31 human leishmaniasis cases were notified through hospital or medical centres in the province of Córdoba in 2023, 24 (77.4%) corresponded to the visceral form, six to the cutaneous (19.4%), and one (3.2%) to the mucocutaneous form. All the 24 cases analysed to species level were caused by L. infantum (Supplementary Material, Fig. 1). Cases were distributed across 15 municipalities, with the municipality of Córdoba (n = 6) and Montilla (n = 5) reporting the highest numbers. Temporal analysis revealed a notable peak in notifications during the autumn and winter months (October to December) (Supplementary Material, Fig. 2). The temporal data on symptoms onset showed no clear tendencies (Supplementary Material, Fig. 3).

Fig. 3.

Seasonal dynamics of sand flies captured every two weeks with CO₂₋baited traps at four sampling sites in Córdoba province (Andalucía, southern Spain) from June to November 2023. (A) Total number of sand flies (all species combined) captured per month; females were not identified to species level, (B) Males of each species captured along the study period. Damaged specimens were not included.

3.2. Seasonal dynamics of sand fly abundance and species composition

An estimated 22,220 sand fly specimens (13,017 females and 9203 males) were captured during the six-month survey conducted at four sampling sites in 2023 (Table 1). Regarding sand fly abundance across the four sampling sites, Aguilar de la Frontera showed the lowest numbers, followed by Córdoba, while the highest abundances were recorded at the other two sites (Table 1).

Table 1.

Counts of female and male sand flies collected at four municipalities in Córdoba (Spain) between June and November 2023.

	June	July	August	September	October	November	Total
Aguilar de la frontera	12	104	74	162	32	0	384
Córdoba	266	228	1381	980	65	0	2920
Fernán Núñez	286	1199	3077	4257	185	0	9004
Montalbán de Córdoba	147	4130	570	4395	666	4	9912
Total	711	5661	5102	9794	948	4	22,220

Based on male morphological identification, Ph. perniciosus was by far the predominant species (n = 8680; 93.4%), followed by Sergentomyia minuta (n = 393; 4.3%), Ph. ariasi (n = 8; 0.08%), and Ph. papatasi (n = 3; 0.005%) (Table 2). Male sand fly collections peaked in September, although captures were already high during July and August. A sharp decline was observed in October, followed by only marginal collections in November (Fig. 3A). While Ph. perniciosus peaked in September, Se. minuta reached its maximum abundance one month earlier, in August. Similar seasonal patterns were observed for females (Fig. 3B).

Table 2.

Counts of male sand flies identified at species level collected at four municipalities in Córdoba (Spain) between June and November 2023.

Species	Aguilar de la Frontera	Córdoba	Fernán Núñez	Montalbán de Córdoba	Total
Ph. perniciosus	103	1255	2814	4508	8680
Se. minuta	8	339	41	5	393
Ph. ariasi	8	0	0	0	8
Ph. papatasi	0	0	0	2	3
Undetermined	17	0	67	35	119
Total	136	1595	2922	4550	9203

3.3. Leishmania detection in sand flies

A total of 134 pools of Phlebotomus spp. and 11 pools of Se. minuta were tested for Leishmania infection. Among these, 107 pools were positive for Leishmania kDNA, resulting in an overall infection prevalence of 3.8%. Of these, 93 pools also yielded a positive ITS1 band, confirming infection with L. infantum. The prevalence of infection per site ranged from 3.4% in Fernán Núñez to 7.5% in Córdoba (Table 3). Specifically, Montalbán de Córdoba showed a significantly higher Leishmania prevalence compared to the other municipalities (p ≤ 0.001; SE = 0.346; Estimate = 1.166). Infection prevalence showed significant differences between June and August (Fig. 4). All the pools tested in October and November were positive for Leishmania.

Table 3.

Summary of infection prevalence of ITS1 positives for L.infantum. For each locality, the prevalence of infection estimated by Maximum Likelihood, the 95% credible interval and the number of Leishmania positive pools out of the number of pools and females tested are given.

Locality	Infection prevalence	Confidence interval	Positive pools/pools tested (females tested)
Aguilar de la frontera	7,3	0,015-0,255	3/4 (110)
Córdoba	7,5	0,040-0,141	18/19 (749)
Fernán Nuñez	3,4	0,023-0,048	36/45 (2156)
Montalbán de Córdoba	7,1	0,044-0,117	36/39 (1754)

Fig. 4.

Estimated monthly infection prevalence from June to September 2023 based on pooled sample testing. Bars indicate the Bayesian point estimates of prevalence, and error bars represent 95% credible intervals. Different letters indicate statistically significant differences. October and November are excluded because all pools tested positive during those months, resulting in extremely wide credible intervals and unreliable estimates.

3.4. Blood meal analysis and host identification

A total of 338 blood-fed female sand flies were analysed, with an overall amplification success rate of 63.9%. Amplification efficiency varied according to both the colour and the volume of blood meal. Specimens with fresh red blood showed a significantly higher amplification success (66.2%) compared to those with black (26.0%) or mixed colour blood (7.6%), χ2 = 55.14, df = 2, p ≤ 0.001). We also found a significantly higher amplification yield in blood-engorged specimens containing a moderate to full amount of blood (χ2 = 14.04, df = 5, p = 0.015) (Fig. 5). Significant differences were detected between score II and scores IV and V (p ≤ 0.05), with a marginal difference between score II and score VI (p = 0.060).

Fig. 5.

Percentage of successful identification of blood meal origin according to feeding scale volume (I = with a trace amount of blood and VI = full blood engorged). The differences between Scale I and II and from III to VI are significant (p ≤ 0.05).

DNA barcoding revealed that all engorged females belonged to Ph. perniciosus (n = 336), except for one Ph. ariasi and one Ph. papatasi. The Iberian rabbit (Oryctolagus cuniculus algirus) was the predominant host (n = 163; 78.3% of the amplified sequences), followed by horses (Equus caballus, n = 36; 17.3%). Five other mammalian species were detected at lower frequencies, including two blood meals from humans (Table 4). These represented only 0.9% of all successfully identified blood meals, whereas 99.1% were derived from other animal hosts, mainly rabbits. Additionally, two samples showed homology with Dromaius novaehollandiae, an Australian species often present in zoological private collections.

Table 4.

Origin of blood meal for sand flies collected at four municipalities in Córdoba (Spain) between June and November 2023.

Sampling municipality	N° blood-engorged	Host-amplification (%)	Oryctolagus cuniculus	Equus caballus	Dromaius novaehollandiae	Felis catus	Homo sapiens	Lepus granatensis	Rattus rattus	Undetermined
Aguilar de la Frontera	6	3 (50.0)	3	0	0	0	0	0	0	0
Córdoba	49	38 (77.5)	35	0	0	1	0	0	0	2
Fernán Núñez	112	94 (83.9)	92	0	0	0	0	0	1	1
Montalbán de Córdoba	171	81 (47.3)	33	36	2	1	2	2	0	5
Total	338	216 (63.9)	163	36	2	2	2	2	1	8

¹ All blood-engorged stages (scale I - VI) are included.

² Include samples that could not be identified to species level due to the poor sequence quality or insufficient sequence length.

4. Discussion

This study provides a comprehensive investigation of the recent leishmaniasis outbreak in Córdoba, with a particular focus on both epidemiological and entomological aspects. By analysing human cases alongside sand fly population dynamics, species diversity, seasonal activity, feeding habits, and infection prevalence, our findings offer valuable insights into the local transmission patterns, which were characterized by a predominance of visceral cases.

We recorded an average of 1852 ± 492 (mean ± SE) sand flies per trapping day, representing one of the highest capture rates reported in Spain to date. Unlike most studies on sand flies in Europe, which primarily rely on sticky and CDC light traps [24], we employed carbon dioxide-baited suction traps, which proved highly efficient for sand fly collection.

In our study, Ph. perniciosus was consistently captured throughout the sampling period, with a pronounced peak in September. Capture numbers declined during the autumn months (October and November), indicating a monophasic phenology across all four sampling sites. This pattern contrasts with other studies that reported a secondary peak in early autumn [24,25]. Regarding Se. minuta, our data indicated a peak in August, although the limited number of specimens precluded robust conclusions about its phenology. Similarly, the low capture rates of other sand fly species did not allow for meaningful analysis of their seasonal dynamics.

A key finding of our study is the high L. infantum infection prevalence recorded in Phlebotomus spp., as revealed by molecular analyses of specimens collected in the study area. Reported Leishmania infection prevalence in sand flies varies widely in the literature, typically ranging from a few percent in large field surveys to higher values in small or highly focal samples, with some PCR-based studies reporting up to ∼35% under specific conditions (e.g., nested PCR or small sample sizes). Overall, large population-level estimates in Mediterranean Europe are generally below ∼15% [26,27]. Although infections in sand flies were detected throughout their entire activity period, the highest prevalence of infection was observed in October and November. This temporal pattern is consistent with previous studies [26,28] and suggest that transmission intensifies toward the end of the warm season, following the gradual build-up of vector populations. However, these results should be interpreted cautiously due to the reduced number of pools available late in the season. Therefore, control measures should be implemented at the beginning of the transmission season, in late spring or early summer, to reduce sand fly densities before the infection peak. Such early interventions are crucial to prevent the increase in human cases, as observed during the 2023 outbreak.

The temporal pattern of human case notifications, with a peak in late summer and autumn season, suggests a possible intensification of transmission toward the end of the warm season. However, no clear temporal pattern of symptom onset was observed. This may reflect the long and variable incubation period of visceral leishmaniasis and possible recall bias among patients, which introduce uncertainty and can obscure real epidemiological trends. The long and variable incubation period of leishmaniasis, often lasting weeks to months, may contribute to the apparent temporal mismatch between sand fly activity and the timing of human case notifications.

The analysis of blood meal sources revealed that Ph. perniciosus predominantly feeds on the Iberian rabbit, with occasional blood meals from other mammals, including horses, hares, and humans. This preference suggests that lagomorphs may serve as significant reservoirs for L. infantum in the region, contributing to the maintenance and transmission of the parasite. Previous studies have identified wild rabbits as common reservoir hosts in various Mediterranean areas [29,30]. A recent analysis of Leishmania prevalence in Andalucia reinforced the role of wild lagomorphs (rabbits and hares) as pivotal L. infantum reservoirs, favouring the occurrence of human leishmaniasis at the wildlife-human-domestic interface [7,31]. Although some authors suggested that the abundance of rabbits could divert sand fly bites away from humans [32], our findings indicate that the high densities of Ph. perniciosus and the concurrent increase in human cases suggest that this potential protective effect is not likely to occur in the outbreak area. Interestingly, human DNA was also detected in some sand fly specimens, supporting human-vector contact even in rural sampling areas. This finding underscores the potential risk of leishmaniasis transmission to humans outside urban settings. Additionally, DNA from horses, cats, black rats, and, unexpectedly, emus was identified. The presence of emu DNA, while surprising, can be attributed to the presence of these birds as exotic pets in the region. Host-feeding patterns are strongly influenced by trap placement and host availability, as documented in previous studies [26]. Given that our traps were deployed in rural areas, primarily located away from densely populated urban settings, a high proportion of human blood meals was not expected. This is consistent with the mammophilic but opportunistic feeding behaviour of Ph. perniciosus, which tends to feed on the most readily available vertebrate hosts in the immediate environment [33]. A recent study refers to such species as “anthropportunist”, emphasizing that their contact with humans reflects opportunistic feeding rather than true anthropophily [34].

Additionally, we provide an illustrated guide to assess the blood status (colour) and the volume of ingested blood. This tool may help prioritize samples with higher chances of successful amplification of vertebrate host DNA. Previous research in mosquitoes has shown that both the volume of the initial blood meal and the stage of digestion are critical factors influencing amplification success [35]. Female sand fly physiological status is commonly classified into three broad categories: unfed, fed, and gravid; however, in most cases, only visibly blood-engorged (reddish) females are selected for host identification analyses [36]. Our results demonstrate reasonable amplification efficiency even in individuals with minimal blood traces. Therefore, incorporating refined visual assessment criteria into protocols may improve host detection rates, particularly when preservation or storage conditions are suboptimal, significantly reducing the costs of blood meal identification studies by focusing on samples with the higher success rate.

A limitation of the study is that we cannot guarantee that all analysed samples contained exclusively females of Ph. perniciosus. Because whole females were homogenized for Leishmania detection, morphological confirmation at the species level was not possible, and potential cryptic diversity could not be assessed. However, the molecular identification of over 330 blood-fed females, almost exclusively corresponded to Ph. perniciosus, provides strong indirect evidence that most of the Phlebotomus spp. females included in the pools also belonged to Ph. perniciosus. This high concordance between male morphological identification and female molecular results supports the reliability of the species composition reported in this study.

The identification of Ph. perniciosus as the most abundant and primary vector, combined with its widespread distribution, is consistent with previous studies across Mediterranean regions. These emphasize the epidemiological relevance of this species in Leishmania transmission [24]. The remaining four sand fly species detected during our survey were present in low numbers, consistent with other reposts from Spain [37], and likely have a minor or negligible impact on Leishmania transmission.

5. Conclusions

Our study highlights the critical role of vector and host dynamics in shaping the risk of leishmaniasis transmission in Córdoba. The exceptionally high densities of Ph. perniciosus and the elevated prevalence of L. infantum infection observed during the outbreak period emphasize the need for continuous entomological surveillance and proactive vector management. Our findings indicate that Ph. perniciosus predominantly feeds on Iberian rabbits, suggesting that this species may act as an important reservoir host in the area. However, since Leishmania infection in rabbits was not directly assessed during this study, further investigations are required to confirm their reservoir status and quantify infection prevalence in the vertebrate reservoirs. The presence of human blood in a few sand fly specimens also reveals a direct human-vector contact, underscoring the potential risk of zoonotic transmission even in rural environments. These results suggest that future surveillance programs should integrate both vector monitoring and reservoir host studies to better understand and manage the transmission dynamics of L. infantum.

Given the marked seasonality of vector activity, intervention should start at the beginning of the season, in late spring, to prevent population build-up and reduce infection risk during summer, when the probability of transmission is highest. Sustainable prevention of human leishmaniasis requires a multifaceted approach, combining targeted vector control, reservoir management, and community-based awareness initiatives. Such integrated strategies, aligned with One Health principles, are essential not only to mitigate outbreaks in Córdoba but also to anticipate and reduce the impact of leishmaniasis in other endemic regions across southern Europe.

Funding

This study has been funded by Consejería de Salud y Consumo de la Junta de Andalucía and Fundación “La Caixa” through the project ARBOPREVENT (HR22–00123). Dra. Nuria Labiod report support from PLEC2021–007968 project (NEXTHREAT) funded by MCIN/AEI/10.13039/5011000110333 and supported by European Union “NextGenerationEU”/PRTR” funds. Grant CEX2024-001498-S funded by MICIU/AEI/10.13039/501100011033.

CRediT authorship contribution statement

Mikel Alexander González: Writing – review & editing, Writing – original draft, Data curation. Nuria Labiod: Writing – review & editing, Methodology, Formal analysis. Sergio Magallanes: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. Maribel Jiménez: Writing – review & editing, Methodology, Formal analysis. Carolina Sanchez-Peña: Writing – review & editing, Funding acquisition. Inés Martín-Martín: Writing – review & editing, Methodology, Formal analysis. Isabel Vázquez-Rincon: Writing – review & editing, Funding acquisition. David Macias-Magro: Writing – review & editing, Funding acquisition. Laura Guillén-Calvo: Writing – review & editing, Methodology, Formal analysis. María José Ruiz López: Writing – review & editing, Methodology, Formal analysis, Data curation. Ana Vázquez: Writing – review & editing, Methodology, Funding acquisition, Formal analysis. Jordi Figuerola: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.