Manuel Morales-Yuste

Introduction: Toscana virus (TOSV) and Leishmania species are sand fly‐borne pathogens that pose a significant public health threat and an increasing risk of exposure in Europe. The objective of this study was to identify the phleboviruses circulating in an area endemic for sand fly‐borne pathogens and to estimate infection prevalence in wild sand fly populations. We also aimed to analyse co‐infections with L. infantum and the symbiotic bacterium Wolbachia . Methods: The study was conducted in southern Spain. Sand flies were collected using CDC light traps, morphologically identified and DNA/RNA was extracted individually or in pools. A generic RT‐nested PCR was used to detect phleboviruses, followed by sequencing and phylogenetic analysis. Leishmania infantum loads were quantified by qPCR, and Wolbachia was detected by PCR. Results: We found that 2/443 individual sand flies (0.45%) and 8/64 pools (12.5%) were infected with phleboviruses, with an estimated prevalence of 1.9%. We confirm the circulation of TOSV‐lineage B, together with Massilia‐like virus and Alcube virus, in three sand fly species, and report novel strains of these phleboviruses. In addition, two Phlebotomus perniciosus individuals were co‐infected with TOSV‐lineage B and Wolbachia wPrn. We also detected L. infantum in 27.6% (60/217) of individuals, 66.7% (40/60) of which were co‐infected with Wolbachia . Mean L. infantum loads [log‐(parasite load +1)] in specimens co‐infected with Wolbachia were not significantly different from those in uninfected specimens ( p = 0.4). Conclusions: This study provides further evidence for the continued circulation of TOSV and other phleboviruses in sand fly populations in southern Spain, with an estimated prevalence higher than that reported in the same region over a decade ago. Our study provides the first evidence of co‐infection with TOSV‐lineage B and Wolbachia in P. perniciosus , as well as a high proportion of specimens co‐infected with Wolbachia and L. infantum . Understanding these co‐infection dynamics is essential to elucidate the potential role of Wolbachia and the impact of Leishmania –phlebovirus co‐infection on 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.

Background: Leishmaniasis, a vector-borne disease caused by the parasite Leishmania, is a public health concern in Europe. Although Spain is an endemic country, it lacks a national surveillance network. To address this, the creation of a research and surveillance network is proposed, which would integrate data from various fields and enhance control and public health strategies. Objectives: The first objective is to gather epidemiological data on vectors, reservoirs, and transmission rates in Spain, with a particular focus on the role of dogs and wildlife as primary reservoirs. The plan includes establishing forums, databases, and specialised training programmes. The second objective is to improve case surveillance and reporting by establishing a comprehensive national registry. Implementing molecular diagnostics and bioinformatics tools will aid analysis of retrospective data and tracking of disease evolution. The third objective is to develop a standardised method for the molecular characterisation of Leishmania, using techniques such as PCR and genomic sequencing to detect virulence and resistance patterns. Key actions include collecting isolates, sharing analytical tools and providing genomic training. The fourth objective is to identify new therapeutic targets in response to toxicity and resistance to current treatments by studying specific enzymes (FeSOD, HDAC), metabolic pathways, and natural products. Ultimately, establishing the Leishmaniasis Surveillance Network will support a “One Health” approach and promote interdisciplinary collaboration towards a national control plan. Methods: We created the Working Group on Leishmaniasis funded by the Spanish Society of Tropical Medicine and International Health as a leishmaniasis forum for meetings and discussions on weaknesses and gaps we find in the knowledge and management of the infection based on the background of each academic and healthcare research group. Expected Impact: This study will promote the articulation of a Leishmaniasis Surveillance Network aiming to bring together the main stakeholders in the research and management of leishmaniasis in Spain.

Leishmaniasis, a vector-borne disease caused by the parasite Leishmania, is a public health concern in Europe. Although Spain is an endemic country, it lacks a national surveillance network. To address this, the creation of a research and surveillance network is proposed, which would integrate data from various fields and enhance control and public health strategies. The first objective is to gather epidemiological data on vectors, reservoirs and transmission rates in Spain, with a particular focus on the role of dogs and wildlife as primary reservoirs. The plan includes establishing forums, databases, and specialised training programmes. The second objective is to improve case surveillance and reporting by establishing a comprehensive national registry. Implementing molecular diagnostics and bioinformatics tools will aid analysis of retrospective data and tracking of disease evolution. The third objective is to develop a standardised method for the molecular char-acterisation of Leishmania, using techniques such as PCR and genomic sequencing to de-tect virulence and resistance patterns. Key actions include collecting isolates, sharing an-alytical tools and providing genomic training. The fourth objective is to identify new therapeutic targets in response to toxicity and resistance to current treatments by studying specific enzymes (FeSOD, HDAC), metabolic pathways and natural products. Ultimately, establishing the Leishmaniasis Surveillance Network will support a 'One Health' ap-proach and promote interdisciplinary collaboration towards a national control plan.

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.

A survey was conducted to assess the impact of on-board gutting in the pouting fishery, Trisopterus luscus (L.), from the Bay of Biscay (area FAO 27.VIII) on the parasite burden of macroscopic ascaridoid nematodes, including anisakids (causing anisakidosis) and raphidascaridids (causing consumer rejection) in these fish. The fish were caught in the Bay of Biscay and collected from the fish market in Granada (southern Spain). Fish larger than 25 cm were gutted on board after capture. A detailed examination of the fish revealed the presence of nematode larvae, which were identified morphologically and molecularly (PCR-RFLP: polymerase chain reaction with restriction fragment polymorphism). Results revealed that ungutted fish harbored only third-stage larvae of ascaridoids (Anisakis and Hysterothylacium) while prevalence reached up to 91%. In contrast, gutted fish exhibited a significant reduction in both the prevalence (36%) and mean abundance (MA, 4.44 vs. 0.91) of these larvae. The prevalence of Anisakis spp. larvae was reduced by over 20%, with a more pronounced reduction in abundance of more than 40% (MA, 1.56 vs. 0.91). Hysterothylacium larvae were completely absent (MA 2.88 vs. 0.00). These findings indicate that gutting, while not highly efficient, lowers Anisakis larvae presence, thereby reducing the risk of anisakiasis to consumers. Additionally, the complete removal of Hysterothylacium larvae enhances the fish’s appearance, making it more appealing and increasing its commercial value, as well as reducing the risk of seizure by health authorities. Further research on these on-board evisceration practices is needed to enhance effectiveness and reduce zoonotic nematodes in commercial fishes.

In order to know whether there is a risk of anisakiasis (or anisakidosis) by consumption of fish of the genus Mullus from the western Mediterranean Sea, which are appreciated for their quality, an epidemiological survey was carried out to evaluate the occurrence of zoonotic or potentially zoonotic nematodes in M. barbatus and M. surmuletus. Although the presence of the third larval stage (L3) of anisakids (Anisakis and Contracaecum) has been previously described in these fish, the results showed the absence of anisakids and the presence, never in muscle, of L3 and L4 of raphidascaridids of the genus Hysterothylacium, molecularly identified as H. fabri. Phylogenetic analysis groups them into the Mediterranean Sea clade, far from individuals isolated in the Pacific Ocean. Prevalence was slightly higher, but not significant, in M. barbatus versus M. surmuletus (72.3% vs 60.0%), but mean intensity (MI) and mean abundance (MA) parameters were approximately twice as high in M. barbatus as in M. surmuletus (MI 5.8 vs 2.8, p = .001; MA 4.2 vs 1.7, p < .001). The presence of the parasite seems to have different effects on these two sympatric species. In M. barbatus it seems to affect their growth, as it appreciably reduces the value of allometry coefficient in infected fish (2.78 vs. 2.18). On the other hand, in M. surmuletus the infection significantly (p < .04) affects the Fulton's condition factor, an indicator of the health status of the fish. It can be concluded that the ingestion of these fish by the people poses negligible risk of anisakiasis, but the consumer should continue to be urged to follow the rules of prevention against this illness.

Parasitism as a lifestyle is much more common in nature than it seems