The epidemiology of feline vector-borne pathogens (FeVBPs) has been less investigated in cats than in dogs. The present study assessed the prevalence of Rickettsia spp., Babesia spp., Cytauxzoon spp. and Leishmania infantum infections in cat populations living in central Italy, by molecular and serological tools. A total of 286 healthy cats were randomly selected from catteries and colonies in central Italy. Peripheral blood and conjunctival swab (CS) samples were collected during surgical procedures for regional neutering projects. Sera were analysed by IFAT to detect anti-Rickettsia felis, R. conorii, Babesia microti and Leishmania IgG antibodies using commercial and home-made antigens. DNA extracted from buffy coats (BCs) was tested for Rickettsia spp., and Piroplasmida species, including Cytauxzoon spp. and Babesia spp. by PCR. Buffy coats and CS samples were assayed by a nested (n)-PCR for Leishmania spp. Sixty-two cats (21.67%) were seropositive to at least one of the tested pathogens. The serological assay revealed 23 (8.04%) and 18 (6.29%) positive cats for R. felis and R. conorii, respectively, with low titers (1/64–1/128). No antibodies against B. microti were detected. Neither Rickettsia nor Piroplasmida DNA were amplified using the specific PCR assays. Thirty-one cats (10.83%) tested positive to anti-Leishmania IgG, with titers ranging from 1:40 to 1:160 and 45 animals (15.73%) tested positive to Leishmania CS n-PCR, whereas none of the animals tested positive to BC n-PCR. Considering the results obtained by IFAT and CS n-PCR, a moderate agreement between the two tests was detected (κ = 0.27). The results of the serological and molecular surveys showed a moderate exposure to Leishmania in the investigated cats and highlighted the limited molecular diagnostic value of BC versus CS samples for this pathogen. Conversely no evidence supported the circulation of Cytauxzoon spp. in domestic cats, in contrast with previous detections in European wild cats in the same areas monitored. The low positive titres for R. felis in association with no DNA BC amplification prevent speculation on the exposure of feline populations to this FeVBP due to the cross-reactivity existing within spotted fever group rickettsiosis (SFGR).

Leishmaniasis in Serbia was an endemic disease, and is considered to be eradicated for more than 40 years. In the past decade sporadic cases of canine leishmaniasis started to emerge for the first time in Vojvodina Province (previously non-endemic region of Serbia). Reports of introduced, and later on autochthonous cases of leishmaniasis alerted the possibility of disease emergence. The aim of this study was to bridge more than a half a century wide gap in entomological surveillance of sand fly vectors in Vojvodina, as well as to verify the presence of the vector species that could support Leishmania spp. circulation. During the period 2013–2015, a total of 136 sand flies were collected from 48 of 80 surveyed locations. Four sand fly species of the genus Phlebotomus were detected: P. papatasi, P. perfiliewi, P. mascittii and P. neglectus. Detection of P. mascittii represents the first record of this species for the sand fly fauna in Vojvodina and in Serbia. All female specimens (n = 80) were tested for Leishmania spp. DNA, and three blood-fed P. papatasi specimens were positive (4%). One positive DNA sample was successfully amplified by ITS1 nPCR. The RFLP analysis of the resulting 350 bp fragment showed a typical pattern of L. infantum, and the ITS1 partial sequence blasted in GenBank confirmed 100% identity with L. infantum and L. donovani complex sequences. This result represents the first record of both Leishmania spp. and L. infantum DNA from sand flies in Vojvodina, and in Serbia. Presence of autochthonous canine leishmaniasis cases, records of Phlebotomus (Larroussius) species proven vectors of L. infantum (P. perfiliewi and P. neglectus) and detection of L. infantum DNA from wild caught (non-competent) vectors, prove that L. infantum is present in Vojvodina and indicates a probable circulation in the region.

Visceral leishmaniasis (VL) is a severe disease caused by Leishmania infantum in the Mediterranean basin, and is associated with considerable morbidity and mortality. Infantile VL may begin suddenly, with high fever and vomiting, or insidiously, with irregular daily fever, anorexia, and marked splenomegaly. Delays in diagnosis of VL are common, highlighting the need for increased awareness of clinicians for VL in endemic European countries. We report 4 cases of young children in northern Italy presenting with persistent fever of unknown origin and diagnosed with VL by serological and molecular methods. At the time of diagnosis, these patients showed an unusual echographic pattern characterized by multiple iso-hypoechoic nodules associated with splenomegaly. We suggest that detection of spleen nodules represents a signature of VL in infants, thus helping to diagnose systemic Leishmania infantum infection in children.

We report a visceral leishmaniasis case in an immunocompetent immigrant with acute renal failure. Parasites were demonstrated in bone marrow, periphe

Background: Drug-resistant strains of Leishmania infantum challenge the effectiveness of treatments for clinical leishmaniosis and may lead to more frequent relapses. Copy number variation (CNV) at specific genetic loci is associated with drug resistance and virulence, but information about its prevalence in endemic regions is limited. This study examines the drug resistance and virulence status of Leishmania strains in human and canine isolates from the Mediterranean region. Methods: Forty-eight Leishmania infantum isolates were whole-genome sequenced with nanopore long reads, followed by de novo assembly. We analyzed chromosomal aneuploidies and gene copy number variation in loci linked to drug resistance and virulence in Leishmania , alongside the genomic structure and rearrangements responsible for these variations. Results: Complete genomes were de novo assembled for 35 L. infantum isolates (22 from dogs and 13 from humans), revealing significant chromosomal variability. We assessed copy number variation for 22 potential biomarkers: 15 genes related to drug resistance to first-line drugs (METK for allopurinol; LdSMT for amphotericin B; AQP1 and H-locus for antimonials; LdMT, LdRos3, and MSL for miltefosine; and PPM for paramomycin) and 7 genes related to virulence (lipophosphoglycan and proteophosphoglycan biosynthesis, and the Lack protein). Drug-resistance biomarkers were identified in 80% of the isolates. Canine strains primarily showed resistance to allopurinol and antimonials, while human isolates exhibited a broader resistance spectrum, especially to antimonials and paromomycin. The co-occurrence of resistance biomarkers was common, especially for allopurinol and antimonial resistance. Distinct mechanisms underlie the observed copy number variations. Virulence-associated genes were less variable among isolates. Conclusions: The prevalence of drug-resistance biomarkers in Leishmania infantum strains from the Mediterranean region, as revealed by this study, underscores the critical need for routine resistance surveillance in managing clinical leishmaniosis. These findings not only inform current clinical practice but also pave the way for more effective management strategies in the future. Graphical

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.