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).

Nematode parasites of the genus Trichinella are important foodborne pathogens transmitted by ingestion of striated muscles harbouring infective larvae. Wild carnivorous and omnivorous animals are the most important reservoirs of these parasites. Hunting activities play an important role in Trichinella spp. epidemiology. The aim of the present work was to assess if serological detection of anti-Trichinella IgG in hunting dogs can be a tool to indirectly monitor Trichinella spp. infections in wildlife. An ELISA and a Western blot (Wb) were developed and validated. To validate the assays, serum samples were collected from 598 dogs considered to be Trichinella-free, 15 naturally infected dogs, and six experimentally infected foxes. Sera were tested by ELISA with Trichinella spiralis excretory/secretory antigens. The diagnostic sensitivity and specificity of ELISA were 100 % (95 % CI: 83.89–100 %) and 95.65 % (95 % CI: 93.69–97.14 %), respectively. Sera from Trichinella-infected dogs/foxes tested by Wb showed a three-band pattern ranging from 48 to 72 kDa. Since the prevalence of Toxocara canis is very high in dogs, the specificity of the ELISA and Wb was further assessed by testing sera for anti-T. canis IgG using T. canis excretory/secretory antigens. No cross-reactivity was observed. To evaluate the test’s reliability in the field, serum samples were collected from wild boar hunting dogs from Central Italy where Trichinella britovi was circulating among wildlife. Out of 384 hunting dog sera, 189 (49.2 %) tested positive by ELISA and of these, 56 (29.6 %) tested positive by Wb, showing an overall prevalence of 14.6 % (56/384) in the wild boar hunting dog population of the investigated area. The serological prevalence in hunting dogs was significantly (P < 0.001) associated with the hunting district’s altitude. This is in agreement with previous investigations, which had shown that the prevalence of T. britovi in wildlife was higher in mountainous areas than in lowland areas of Italy. The results suggest that the circulation of Trichinella spp. among wildlife can be monitored by testing sera from hunting dogs, which could act as sentinel animals of Trichinella spp. circulation in wildlife.

Background: Among pathogenic Leishmania species, L. major and L. tropica , occur mainly in North African and Middle Eastern countries, primarily transmitted by Phlebotomus papatasi and P. sergenti respectively. In Italy, P. perfiliewi is the second most abundant sand fly species involved in L. infantum transmission exhibiting opportunistic behaviour in rural and semi-urban settings. Experimental infections were conducted to assess the susceptibility of a wild Italian P. perfiliewi population to the development of L. tropica and L. major . Methods: Experiments were performed with a wild P. perfiliewi population, collected in Magliano in Toscana (Tuscany, Italy). In separate experiments, females were artificially infected with a suspension of L. tropica or L. major (10 ⁶ promastigotes/ml). Infection progression was monitored through microscopic midgut dissection and supported by molecular analyses. Results: Experimental infections with L. tropica yielded 5923 engorged females, with an average feeding rate of 25.7% and a survival rate of 70%. The infection positivity rate was 25.2%, significantly lower than that observed in the control species. Nonetheless, parasite migration to the stomodeal valve suggests that P. perfiliewi may support the full developmental cycle of L. tropica , although less efficiently than the control species. Infections with L. major showed a significantly lower mean feeding rate ( p = 0.05) and a positivity rate of 46.6%, compared to 70.5% in P. perniciosus ( p = 0.03). Still, P. perfiliewi appeared capable of supporting L. major metacyclogenesis, even under light to moderate parasite loads ( p = 0.007). Conclusions: These findings suggest that P. perfiliewi can support the development of both L. tropica and L. major under experimental conditions, with greater susceptibility observed for L. tropica . However, without in vivo confirmation or xenodiagnosis, its capacity for natural transmission remains unestablished. Nonetheless, implementing integrated One Health surveillance strategies would be advisable to monitor potential changes in the epidemiology of both exotic and endemic Leishmania species. Graphical

L. infantum and L. donovani, distinct species in the L. donovani complex, show high phenotypic and genotypic polymorphism and share molecular traits. Therefore, genotyping by a single molecular target can give uncertain results. This study focuses on genotyping a set of L. donovani complex strains, including 18 zymodemes classified according to Montpellier nomenclature (ZMONs) and different clinical forms, by internal transcribed spacer (ITS), heat shock protein 70 (hsp70), and cysteine proteinase b (cpb) sequencing to evaluate their ability in species discrimination. We found an unexpected L. infantum hsp70 variability, with 8 sequence variants. Cpb-PCR could not distinguish L. donovani complex species, due to a L. infantum intraspecific allelic (cpbEF) polymorphism. By combining ITS-hsp70-cpb sequence variants, we obtained different genotypes. ITS(A)-hsp70inf(2)-cpbE identified 69.9% of L. infantum strains representing 12 ZMONs from Mediterranean visceral and cutaneous cases, ITS(A)-hsp70inf(2)-cpbF identified a non-ZMON-1 cluster. Four genotypes represented ZMON-24: ITS(A, B)-hsp70(Y)-cpbF identified a cutaneous cluster from Italy and North Africa, ITS(A, Lombardi)-hsp70(2)-cpbE identified cutaneous and visceral cases from Mediterranean areas. We believe this study contributes to an overview of L. infantum variant populations, and to the discussion on diagnostic targets, in single or multi -target-based approaches, to identify Leishmania populations in the Mediterranean area.

Leishmania infantum is the primary causative agent of zoonotic leishmaniasis in Europe and is transmitted by phlebotomine sand flies. Although Slovenia is not considered endemic, competent vectors and canine infections have been documented, suggesting conditions that may allow local transmission. During the 2023 sand fly season, 145 phlebotomine sand flies were collected at a farm site in Cetore, Slovenian Istria, and identified morphologically as Phlebotomus neglectus , P . perniciosus and P . mascittii . Female sand flies were grouped into eight monospecific pools and screened for Leishmania DNA by kinetoplast DNA real‐time PCR. One pool of P . neglectus females collected in July tested positive, with a low cycle threshold value indicating a high parasite DNA load. Species‐level identification using ITS‐1 and hsp70 PCR‐RFLP, followed by sequencing, confirmed the parasite as L . infantum . This study provides the first molecular confirmation of L . infantum in phlebotomine sand flies from Slovenia. The finding of L . infantum DNA in a proven vector species represents an early warning signal and highlights the need to implement entomological and molecular surveillance programmes in Slovenia.