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Leptospirosis is a bacterial disease of worldwide distribution with relevant implications for animal and human health. Different large wild carnivore species can act as reservoirs of this zoonotic pathogen. This study aimed to evaluate the circulation of Leptospira spp. in free-ranging wolves (Canis lupus) from southern Europe. A total of 281 kidney samples of wolves from Spain and Italy were collected between 2017 and 2023. The presence of Leptospira DNA was analysed by real-time PCR and phylogenetic analyses were carried out using a Bayesian approach. The overall prevalence was 3.2 % (9/281; 95 %CI: 1.1-5.3). Leptospira DNA was detected in nine of the 180 wolves from Spain (5.0 %; 95 %CI: 1.8-8.2), but not in the Italian wolf population (0 %; 0/101). Molecular analyses revealed high homology between the sequences obtained in the present study and isolates of Leptospira interrogans and Leptospira borgpetersenii from different rodent and domestic ungulate species. Our results provide evidence of a low and spatially heterogeneous circulation of this pathogen in wolf populations of southern Europe. The detection of zoonotic Leptospira species in this survey supports the need to consider wolf populations in monitoring programs for leptospirosis with a One Health approach., Competing Interests: Declaration of Competing Interest None of the authors of this study has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Leptospirosis is a re-emerging and globally spread zoonosis caused by pathogenic genomospecies of Leptospira. Wild boar (Sus scrofa) are an important Leptospira host and are increasing in population all over Europe. The aim of this investigation was to evaluate Leptospira spp. infection in the reproductive systems of wild boar hunted in two Italian regions: Tuscany and Sardinia. From 231 animals, reproductive system tissue samples (testicles, epididymides, uteri) as well as placentas and fetuses were collected. Bacteriological examination and Real-Time PCR were performed to detect pathogenic Leptospira (lipL32 gene). Leptospires were isolated from the testicles and epididymides of one adult and two subadult wild boar. Four isolates from the two subadult males were identified as Leptospira interrogans serogroup Australis by MLST, whereas Leptospira kirschneri serogroup Grippotyphosa was identified from the adult testicles and epididymis. Using Real-Time PCR, 70 samples were positive: 22 testicles (23.16%) and 22 epididymides (23.16%), 10 uteri (7.35%), 3 placentas (6.66%), and 13 fetuses (28.88%). Amplification of the rrs2 gene identified L. interrogans and L. kirschneri species. The results from this investigation confirmed that wild boar represent a potential source of pathogenic Leptospira spp. Isolation of Leptospira serogroups Australis and Grippotyphosa from the male reproductive system and the positive Real-Time PCR results from both male and female samples could suggest venereal transmission, as already demonstrated in pigs. Furthermore, placentas and fetuses were positive for the lipL32 target, and this finding may be related to a possible vertical transmission of pathogenic Leptospira., Competing Interests: The authors have declared that no competing interests exist.

The authors describe work in progress at the laboratory in Brescia, Italy, on the application of molecular methods to the diagnosis of leptospirosis. This work includes the following: a) Development of polymerase chain reaction (PCR) assays capable of amplifying specific deoxyribonucleic acid fragments from most Leptospira interrogans strains. b) Development of a microtitre-based assay for rapid detection of PCR-positive samples. c) Characterisation of Leptospira strains through restriction endonuclease analysis of PCR products and amplified fragment length polymorphism.