Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of paratuberculosis. The aim of our study was to combine Mini-and Microsatellite loci analysis in order to explore the effectiveness of this sub-typing method in a group of Map isolates. For this purpose, 84 Italian Type C Map isolates, each from a different cattle herd, were submitted to MIRU-Variable-Number Tandem-Repeats (VNTRs) typing and Short Sequence repeats (SSRs) sequencing. Moreover, the method was used to analyse the variability inside 10 herds (from three to 50 isolates per herd).

Sand flies transmit Leishmania infantum, which is responsible for causing leishmaniasis, as well as many phleboviruses, including the human pathogenic Toscana virus. We screened sand flies collected from a single site between 2017 and 2020 for the presence of both phleboviruses and Leishmania. The sand flies were sampled with attractive carbon dioxide traps and CDC light traps between May and October. We collected more than 50,000 sand flies; 2826 were identified at the species level as Phlebotomus perfiliewi (98%) or Phlebotomus perniciosus (2%). A total of 16,789 sand flies were tested in 355 pools, and phleboviruses were found in 61 pools (6 Toscana virus positive pools, 2 Corfou virus positive pools, 42 Fermo virus positive pools, and 7 Ponticelli virus positive pools, and 4 unidentified phlebovirus positive pools). Leishmania was found in 75 pools and both microorganisms were detected in 16 pools. We isolated nine phleboviruses from another 2960 sand flies (five Ponticelli viruses and for Fermo viruses), not tested for Leishmania; the complete genome of a Fermo virus isolate was sequenced. The simultaneous detection in space and time of the Fermo virus and L. infantum is evidence that supports the co-circulation of both microorganisms in the same location and partial overlap of their cycles. A detailed characterization of the epidemiology of these microorganisms will support measures to limit their transmission.

In the last decade, an upsurge of human leishmaniasis has been reported in the Emilia-Romagna region, Northeast Italy. Epidemiologic data have raised doubts about the role of dogs as the main reservoirs for Leishmania infantum. In the present study, a total of 1077 wild animals were screened for L. infantum DNA in earlobe and spleen samples from 2019 to 2022. The lymph nodes were tested only in 23 animals already positive in the earlobe and/or spleen. A total of 71 (6.6%) animals resulted positive in at least one of the sampled tissues, including 3/18 (16.7%) wolves, 6/39 (15.4%) European hares, 38/309 (12.3%) roe deer, 1/11 (9.1%) red deer, 8/146 (4.9%) wild boars, 13/319 (4.1%) red foxes, 1/54 (1.9%) porcupine, and 1/59 (1.7%) European badger. Most of the infected animals (62/71) tested positive only in the earlobe tissue, only four animals (two roe deer and two wild boars) tested positive only in the spleen, and five animals (three roe deer and two red foxes) resulted positive for both tissues. L. infantum DNA was detected in the lymph nodes of 6/23 animals. L. infantum detection occurred in all seasons associated with low real-time PCR Ct values. Further research is needed in order to clarify the role of wildlife in the re-emerging focus of leishmaniasis in Northeast Italy.

Animals, including wildlife, are part of One-Health concept since many infectious diseases can affect both humans and animals. In this study, 126 red foxes (Vulpes vulpes) from Northern Italy in 2022–2023 were tested by molecular assays for Protoparvovirus carnivoran 1 (PPVC-1), Canine adenovirus type 1 and 2 (CAdV-1 and CAdV-2), Circovirus canine (CanineCV), Canine distemper virus (CDV), and Leptospira spp. A total of 39 of 126 (30.9%) red foxes were infected with at least one pathogen and five of these were coinfected: 20/126 (15.9%) red foxes tested positive for PPVC-1, 3/126 (2.4%) for CAdV, 20/126 (15.9%) for CanineCV, and 2/126 (1.6%) for Leptospira spp. DNA. No foxes tested positive for CDV RNA. The pathogens identified were genetically analysed. New findings were reported such as a fox with multiple feline panleukopenia virus (FPV) and canine parvovirus type 2b (CPV-2b) infection associated with quasispecies dynamics, typical genetic characteristics of the identified CanineCV, and the first detection in red foxes of Leptospira ST198 related to L. interrogans serogroup Australis. Further studies are necessary to investigate the transmission between domestic animals and wildlife and to understand the role of red foxes in the maintenance of these pathogens not only in the wild but also in urban and peri-urban environments.

Listeria monocytogenes is an ubiquitous pathogen isolated from different host species including fish, crustaceans, and molluscs, but it is rarely a pathogenic microorganism to marine reptiles. In particular, only two cases of fatal disseminated listeriosis have been described in the loggerhead sea turtle (Caretta caretta). In this study, we describe a lethal case of L. monocytogenes infection in a loggerhead sea turtle. The turtle was found alive, stranded on a beach in North-eastern Italy, but perished soon after being rescued. The autoptic examination revealed that heart, lung, liver, spleen, and urinary bladder were disseminated with multiple, firm, 0.1–0.5 mm sized, nodular, white-green lesions. Microscopically, these lesions corresponded with heterophilic granulomas with Gram+ bacteria within the necrotic center. Furthermore, the Ziehl–Neelsen stain was negative for acid-fast organisms. Colonies isolated from heart and liver were tested through MALDI-TOF for species identification, revealing the presence of L. monocytogenes. Whole Genome Sequencing on L. monocytogenes isolates was performed and the subsequent in silico genotyping revealed the belonging to Sequence Type 6 (ST 6); the virulence profile was evaluated, showing the presence of pathogenicity islands commonly observed in ST 6. Our results further confirm that L. monocytogenes should be posed in differential diagnosis in case of nodular lesions of loggerhead sea turtles; thus, given the zoonotic potential of the microorganism, animals should be treated with particular caution. In addition, wildlife animals can play an active role as carriers of possibly pathogenetic and virulent strains and contribute to the distribution of L. monocytogenes in the environment.

Cases of sand fly-borne diseases in the Emilia-Romagna region, such as meningitis caused by Toscana virus and human leishmaniasis, are reported annually through dedicated surveillance systems. Sand flies are abundant in the hilly part of the region, while the lowland is unsuitable habitat for sand flies, which are found in lower numbers in this environment with respect to the hilly areas. In this study, we retrieved sand flies collected during entomological surveillance of the West Nile virus (from 2018 to 2021) to assess their abundance and screen them for the presence of pathogens. Over the four-year period, we collected 3022 sand flies, more than half in 2021. The most abundant sand fly species was Phlebotomus (Ph.) perfiliewi, followed by Ph. perniciosus; while more rarely sampled species were Ph. papatasi, Ph. mascittii and Sergentomyia minuta. Sand flies were collected from the end of May to the end of September. The pattern of distribution of the species is characterized by an abundant number of Ph. perfiliewi in the eastern part of the region, which then falls to almost none in the western part of the region, while Ph. perniciosus seems more uniformly distributed throughout. We tested more than 1500 female sand flies in 54 pools to detect phleboviruses and Leishmania species using different PCR protocols. Toscana virus and Leishmania infantum, both human pathogens, were detected in 5 pools and 7 pools, respectively. We also detected Fermo virus, a phlebovirus uncharacterized in terms of relevance to public health, in 4 pools. We recorded different sand fly abundance in different seasons in Emilia-Romagna. During the season more favorable for sand flies, we also detected pathogens transmitted by these insects. This finding implies a health risk linked to sand fly-borne pathogens in the surveyed area in lowland, despite being considered a less suitable habitat for sand flies with respect to the hilly areas.