Retrospective immunohistochemical investigation on dolphin morbillivirus infection by comparing the performance of heterologous monoclonal and polyclonal antibodies - Short communication.

Dolphin morbillivirus (DMV) is a pathogen of great concern in free-ranging cetaceans. Confirmation and staging of morbillivirus infections rely on histology and immunohistochemistry (IHC), following molecular detection. As at the present time no specific antibodies (Abs) against DMV are available, two heterologous Abs have been used worldwide for the examinations of morbillivirus infections of cetaceans. One is a monoclonal Ab (MoAb) prepared against the N protein of canine distemper virus (CDV), whereas the other is a polyclonal Ab raised in rabbits against rinderpest virus (RPV). Both Abs are known to show cross-reactivity with DMV. In this study we compared the labelling quality and the neuroanatomical distribution of staining with these two Abs by means of IHC analysis. To this end, serial sections of the target organs from ten free-ranging stranded cetaceans, previously diagnosed as being infected with DMV by PCR and/or serology, were subjected to IHC. The brain, lungs and lymph nodes of one animal were found to be positive with both Abs. From two other animals, the brain and the spleen, respectively, tested positive only with the polyclonal Ab. In the positive brain tissues, multifocal immunostaining was observed, with similar staining location and extent, with the two antibodies tested. Our results suggest that the polyclonal anti-RPV Ab might have a stronger binding activity to DMV than the anti-CDV MoAb. Nevertheless, the elaboration and use of specific anti-DMV Abs might be essential to guarantee conclusive results in diagnostic and pathogenetic investigations.

Evidence for Unknown Sarcocystis-Like Infection in Stranded Striped Dolphins (Stenella coeruleoalba) from the Ligurian Sea, Italy.

Two striped dolphins (SD1, SD2), stranded along the Ligurian coast of Italy, were diagnosed with a nonsuppurative meningoencephalitis associated with previously undescribed protozoan tissue cysts. As tissue cysts were morphologically different from those of Toxoplasma gondii, additional histopathological, immunohistochemical, ultrastructural, and biomolecular investigations were performed, aiming to fully characterize the organism. Histopathology revealed the presence of large Sarcocystis-like tissue cysts, associated with limited inflammatory lesions in all CNS areas studied. IHC was inconclusive, as positive staining with polyclonal antisera did not preclude cross-reaction with other Sarcocystidae coccidia. Applied to each animal, 11 different PCR protocols precluded a neural infection by Sarcocystis neurona, Sarcocystis falcatula, Hammondia hammondi, and Neospora caninum. T. gondii coinfection was confirmed only in dolphin SD2. Sarcocystis sp. sequences, showing the highest homology to species infecting the Bovidae family, were amplified from SD1 myocardium and SD2 skeletal muscle. The present study represents the first report of Sarcocystis-like tissue cysts in the brain of stranded cetaceans along with the first description of Sarcocystis sp. infection in muscle tissue of dolphins from the Mediterranean basin.

Ticks infesting humans and associated pathogens: a cross-sectional study in a 3-year period (2017-2019) in northwest Italy.

BACKGROUND: Tick-borne diseases are common throughout Europe. Ticks transmit pathogens to the host while feeding and together with mosquitoes, they are major vectors of infectious agents worldwide. In recent years, there has been a marked increase in the incidence of tick-bite events and tick-borne disease in northwest Italy, but information on the prevalence of tick-borne pathogens in ticks removed from humans remains scarce. To fill this gap, we report here the prevalence of tick bites and tick-borne pathogens documented for humans in Piedmont, northwest Italy, in the 3-year period 2017-2019. METHODS: Ticks attached to humans during 2017-2019 were collected from residents of urban and rural area by physicians and veterinarians working with local veterinary agencies. All ticks (n = 1290) were morphologically identified to the species level. A subset of ticks removed from children (age 0-18 years) and the elderly (> 70 years), both age groups considered to be at-risk populations, was screened by biomolecular analysis to detect pathogens (e.g. Rickettsia spp., Borrelia spp., Anaplasma spp.). Pathogen identity was confirmed by Sanger sequencing. RESULTS: Ticks were taxonomically assigned to ten species of six genera (Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus). Most belonged to the genus Ixodes: 1009 ticks (78.22%) were classified as Ixodes ricinus. A subset of 500 ticks collected from the two at-risk populations were subjected to PCR assay to determine the presence of Rickettsia spp., Borrelia spp., and Anaplasma spp. The overall prevalence of infection was 22.8% (n = 114; 95% confidence interval [CI]: 19.19-26.73%), meaning that at least one pathogen was detected: Rickettsia spp. (prevalence 15%, n = 76; 95% CI 12.17-18.65%); Borrelia spp. (prevalence 6.4%, n = 32; 95% CI 4.42-8.92%); and Anaplasma spp. (prevalence 1.2%, n = 6; 95% CI 0.44-2.6%). CONCLUSIONS: Our data underline the importance of surveillance in the epidemiology of tick-borne diseases and the implementation of strategies to control tick infestation and associated pathogens.

Occurrence of Brucella ceti in striped dolphins from Italian Seas.

Brucella ceti infections have been increasingly reported in cetaceans, although a very limited characterization of Mediterranean Brucella spp. isolates has been previously reported and relatively few data exist about brucellosis among cetaceans in Italy. To address this gap, we studied 8 cases of B. ceti infection in striped dolphins (Stenella coeruleoalba) stranded along the Italian coastline from 2012 to 2018, investigated thanks to the Italian surveillance activity on stranded cetaceans. We focused on cases of stranding in eastern and western Italian seas, occurred along the Apulia (N = 6), Liguria (N = 1) and Calabria (N = 1) coastlines, through the analysis of gross and microscopic findings, the results of microbiological, biomolecular and serological investigations, as well as the detection of other relevant pathogens. The comparative genomic analysis used whole genome sequences of B. ceti from Italy paired with the publicly available complete genomes. Pathological changes consistent with B. ceti infection were detected in the central nervous system of 7 animals, showing non-suppurative meningoencephalitis. In 4 cases severe coinfections were detected, mostly involving Dolphin Morbillivirus (DMV). The severity of B. ceti-associated lesions supports the role of this microbial agent as a primary neurotropic pathogen for striped dolphins. We classified the 8 isolates into the common sequence type 26 (ST-26). Whole genome SNP analysis showed that the strains from Italy clustered into two genetically distinct clades. The first clade comprised exclusively the isolates from Ionian and Adriatic Seas, while the second one included the strain from the Ligurian Sea and those from the Catalonian coast. Plotting these clades onto the geographic map suggests a link between their phylogeny and topographical distribution. These results represent the first extensive characterization of B. ceti isolated from Italian waters reported to date and show the usefulness of WGS for understanding of the evolution of this emerging pathogen.

First report of the invasive mosquito Aedes koreicus (Diptera: Culicidae) and of its establishment in Liguria, northwest Italy.

BACKGROUND: Invasive mosquito species (IMS) of the genus Aedes are a cause of increasing concern in Europe owing to their ability to vector important human viral diseases. Entomological surveillance to early detect alien mosquito and flavivirus circulation in Liguria, northwest Italy, has been carried out since 2011. RESULTS: The invasive species Aedes koreicus was first detected in Genoa in September 2015, when a male specimen was caught near the international airport; species identity was confirmed by genetic analysis. Over the next three years, 86 more adult specimens were trapped at sites throughout the city, accounting for 0.50% of all mosquitoes and 1.04% of Aedes sp. mosquitoes trapped in Genova in the four-year period 2015-2018. So far, no other monitored sites in Liguria have revealed the presence of this species. Ovitraps at two sites became positive for the species in July-August 2017. All female Ae. koreicus pools analysed were negative in biomolecular assays for Flavivirus. CONCLUSIONS: Our findings of Ae. koreicus in Genoa constitute, to the best of our knowledge, the first report of the species in northwest Italy and in a Mediterranean port city. The species appears to be established; trapping and climatic data support survival of Ae. koreicus in the area through three consecutive winters. Monitoring of adult mosquitoes detected the species two years before its discovery with ovitraps; trapping for adult specimens appears to be a more effective tool for the early detection of IMS. The airport (located near the commercial port area) and the flower market are the most probable sites of introduction; however, the exact time and place of arrival of this IMS in Liguria remain unknown. Based on morphological and genetic data, a common origin for most of the Ae. koreicus populations established in Europe is suspected. So far, no control measures have been adopted in Genoa and the species will probably colonize an even wider area in the next few years.

Novel dolphin morbillivirus (DMV) outbreak among Mediterranean striped dolphins Stenella coeruleoalba in Italian waters.

An unusual mortality event (UME) of striped dolphins Stenella coeruleoalba occurred in the period July to December 2016 along the Italian Ionian coastline. We conducted a complete postmortem examination on 28 specimens and detected dolphin morbillivirus (DMV), by means of biomolecular analyses, in the target tissues of 17 animals. Unlike previous outbreaks occurring in the Mediterranean Sea in 2011 and 2013, we observed typical pathological changes suggestive of morbilliviral infection in an acute/subacute phase and immunohistochemical reactivity. The same findings were observed in 13 other specimens beached along the Italian coastline during 2016 with no temporal and geographical relationship with the ongoing epidemic outbreak. Molecular characterization and phylogenetic analysis showed that DMV sequences detected in Italy in 2016 clustered with those identified in Portugal and Galicia (Spain), representing a novel DMV strain of Atlantic origin which entered the Mediterranean Sea and affected a naïve striped dolphin population. DMV sequences detected in the previous Mediterranean outbreaks exhibited a marked genetic relatedness and diverged from those detected in cetaceans stranded along the Galician and Portuguese coasts since 2007.

Multidisciplinary studies on a sick-leader syndrome-associated mass stranding of sperm whales (Physeter macrocephalus) along the Adriatic coast of Italy.

Mass strandings of sperm whales (Physeter macrocephalus) are rare in the Mediterranean Sea. Nevertheless, in 2014 a pod of 7 specimens stranded alive along the Italian coast of the Central Adriatic Sea: 3 individuals died on the beach after a few hours due to internal damages induced by prolonged recumbency; the remaining 4 whales were refloated after great efforts. All the dead animals were genetically related females; one was pregnant. All the animals were infected by dolphin morbillivirus (DMV) and the pregnant whale was also affected by a severe nephropathy due to a large kidney stone. Other analyses ruled out other possible relevant factors related to weather conditions or human activities. The results of multidisciplinary post-mortem analyses revealed that the 7 sperm whales entered the Adriatic Sea encountering adverse weather conditions and then kept heading northward following the pregnant but sick leader of the pod, thereby reaching the stranding site. DMV infection most likely played a crucial role in impairing the health condition and orientation abilities of the whales. They did not steer back towards deeper waters, but eventually stranded along the Central Adriatic Sea coastline, a real trap for sperm whales.

The diatoms test in veterinary medicine: A pilot study on cetaceans and sea turtles.

Fishing activities are considered one of the most relevant threats for cetaceans and sea turtles conservation since these animals are sometimes found dead entangled in fishing gears. Currently, postmortem diagnosis is based mainly on the presence of nets and lines on the body and the related marks and injuries evident at gross examination. A more detailed and objective evidence is needed to clarify doubts cases and the diatoms technique, used in forensic human medicine, could support drowning diagnosis also in this field. Diatoms' investigation was implemented to be applied in marine vertebrate on 8 striped (Stenella coeruleoalba) and 1 bottlenose (Tursiops truncatus) dolphins and 5 sea turtles (Caretta caretta) stranded along the Italian coastlines with a likely cause of death hypothized on necropsies carried out by veterinary pathologists. Diatoms were microscopically searched in the bone marrow collected from long bones implementing protocols used in human medicine and their presence was observed in 4 cetaceans and 2 sea turtles. Despite a clear relation between diatoms' presence and amount and the likely cause of death was not proved due to the poor number of samples, the higher burden of diatoms was found in 3 animals deemed to be death for the interaction with human activity. Despite more studied are necessary to identify the possible relation between the cause of death and diatoms' findings, the present study implemented this technique to be adapted to marine animals, confirming its possible application also in veterinary forensic medicine.

Analysis of the environmental and host-related factors affecting the distribution of the tick Dermacentor marginatus.

Understanding and responding to the ecological, social and economic conditions that facilitate disease emergence and transmission represents a substantial challenge for epidemiologists and health professionals. In this article we integrate knowledge about the human and the vector population, to provide a context from which to examine the underlying causal factors of D. marginatus-borne diseases emergence in the study area. Within this framework we analyse the biotic and abiotic factors that drive D. marginatus population dynamics and the role of its typical host for dispersal. These investigations suggest that D. marginatus is a tick species prone to spatially overlap its presence with human population presence. Then we consider the public health implications for the residents, when simply carrying out trivial outdoor activities may increase the risk to contact an infected tick.

Occlusive mycotic tracheobronchitis and systemic Alphaherpesvirus coinfection in a free-living striped dolphin Stenella coeruleoalba in Italy.

A juvenile female striped dolphin Stenella coeruleoalba live stranded on 4 March 2016 at Alassio, western Ligurian Sea coast, Italy. The dolphin died shortly after stranding, and a complete postmortem examination was performed. Necropsy revealed severe tracheal occlusion and unilateral bronchial stenosis with luminal accumulation of abundant green-yellow mucous-gelatinous material. Histological features suggestive of tracheobronchial aspergillosis were observed. Cultures of lung tissue and tracheo-bronchial exudate isolated Aspergillus fumigatus, identified by a Microseq D2 LSUrDNA fungal sequencing kit. A pan-Herpesvirus nested-PCR assay on frozen samples obtained from multiple organs was positive. Phylogenetic analysis on the partial DNA polymerase gene revealed that the striped dolphin isolate was closely related to known cetacean Alphaherpesvirus sequences from the same host species. Attempted virus isolation was unsuccessful. The tissue levels of different persistent organic pollutants and the toxicological stress, evaluated using a theoretical model, showed a severely impaired immune response. This study reports the first case of occlusive mycotic tracheobronchitis in a free-living cetacean and the first molecular identification of an Alphaherpesvirus in a free-ranging striped dolphin stranded on the coast of Italy.

Efficient isolation on Vero.DogSLAMtag cells and full genome characterization of Dolphin Morbillivirus (DMV) by next generation sequencing.

The Dolphin Morbillivirus (DMV) genome from the first Mediterranean epidemic (1990-'92) is the only cetacean Morbillivirus that has been completely sequenced. Here, we report the first application of next generation sequencing (NGS) to morbillivirus infection of aquatic mammals. A viral isolate, representative of the 2006-'08 Mediterranean epidemic (DMV_IZSPLV_2008), efficiently grew on Vero.DogSLAMtag cells and was submitted to whole genome characterization by NGS. The final genome length was 15,673 nucleotides, covering 99.82% of the DMV reference genome. Comparison of DMV_IZSPLV_2008 and 1990-'92 DMV strain sequences revealed 157 nucleotide mutations and 47 amino acid changes. The sequence similarity was 98.7% at the full genome level. Whole-genome phylogeny suggested that the DMV strain circulating during the 2006-'08 epidemics emerged from the 1990-'92 DMV strain. Viral isolation is considered the "gold standard" for morbillivirus diagnostics but efficient propagation of infectious virus is difficult to achieve. The successful cell replication of this strain allowed performing NGS directly from the viral RNA, without prior PCR amplification. We therefore provide to the scientific community a second DMV genome, representative of another major outbreak. Interestingly, genome comparison revealed that the neglected L gene encompasses 74% of the genetic diversity and might serve as "hypervariable" target for strain characterization.

NEUROPATHOLOGIC FINDINGS IN CETACEANS STRANDED IN ITALY (2002-14).

We summarized the neuropathologic findings in 60 cetaceans stranded along the Italian coastline from 2002 to 2014. The following neuropathologic changes were detected in 45% (27/60) of animals: nonsuppurative meningo-encephalitides (30%, 18/60), nonspecific lesions (12%, 7/60), suppurative encephalitis (2%, 1/60), and neoplasm (2%, 1/60). No histologic lesions were found in 47% (28/60) of the specimens. Five (8%, 5/60) samples were unsuitable for analysis. Analysis with PCR detected Brucella spp., morbillivirus, and Toxoplasma gondii infection in one, six, and seven individuals, respectively. Immunohistochemical analysis confirmed positivity for morbillivirus and for T. gondii infection in three cases each. No evidence of the scrapie-associated prion protein PrPSc was detected. Our findings underscore the importance of an adequate surveillance system for monitoring aquatic mammal pathologies and for protecting both animal and human health.

The Degree of One Health Implementation in the West Nile Virus Integrated Surveillance in Northern Italy, 2016.

West Nile virus (WNV) is endemic in the Po valley area, Northern Italy, and within the legal framework of the national plan for the surveillance of human vector-borne diseases, WNV surveillance has over time been implemented. The surveillance plans are based on the transdisciplinary and trans-sectorial collaboration between regional institutions involved in public, animal, and environmental health. This integrated surveillance targets mosquitoes, wild birds, humans, and horses and aims at early detecting the viral circulation and reducing the risk of infection in the human populations. The objective of our study was to assess the degree of One Health (OH) implementation (OH-ness) of the WNV surveillance system in three North Italian regions (Emilia-Romagna, Lombardy, Piedmont) in 2016, following the evaluation protocol developed by the Network for Evaluation of One Health (NEOH). In detail, we (i) described the OH initiative (drivers, outcomes) and its system (boundaries, aim, dimensions, actors, stakeholders) and (ii) scored different aspects of this initiative (i.e., OH-thinking, -planning, -sharing, -learning, transdisciplinarity and leadership), with values from 0 (=no OH approach) to 1 (=perfect OH approach). We obtained a mean score for each aspect evaluated. We reached high scores for OH thinking (0.90) and OH planning (0.89). Lower scores were attributed to OH sharing (0.83), transdisciplinarity and leadership (0.77), and OH learning (0.67), highlighting some critical issues related to communication and learning gaps. The strengths and weaknesses detected by the described quantitative evaluation will be investigated in detail by a qualitative evaluation (process evaluation), aiming to provide a basis for the development of shared recommendations to refine the initiative and conduct it in a more OH-oriented perspective.

POSTMORTEM FINDINGS IN CETACEANS FOUND STRANDED IN THE PELAGOS SANCTUARY, ITALY, 2007-14.

Between 2007 and 2014, 83 cetaceans were found stranded along the Ligurian coast of Italy, in the Pelagos Sanctuary, the largest marine protected area in the Mediterranean basin. Forty-nine (59%) were submitted to complete or partial necropsy, depending on the conservation status of the carcass. Based on gross and histological pathology and ancillary testing, the cause of death was determined and categorized as anthropogenic or natural (i.e., nonanthropogenic) in origin for 33 animals (67%) and of undetermined origin in the remaining 16 (33%). Natural causes of death, accompanied by either poor or good nutritional status, were attributed to 29 animals (59%), whereas four (8%) were diagnosed with an anthropogenic cause of death, consisting of interaction with fishing activities. Infectious and noninfectious disease was the most common cause of death, involving 29 cetaceans (59%). These data are valuable for understanding health and mortality trends in cetacean populations and can provide information for establishing policies for cetacean conservation and management in such an important protected area of the Mediterranean basin.

Detection of morbillivirus infection by RT-PCR RFLP analysis in cetaceans and carnivores.

Morbillivirus genus comprises several members related to specific hosts, such as canine distemper virus (CDV) and cetacean morbillivirus (CeMV) in which the dolphin morbillivirus (DMV) is included. Both CDV and DMV are able to cause serious outbreak associated with high morbidity and mortality representing an important conservation threat for terrestrial and aquatic mammalian species. This paper describes a new RT-PCR RFLP technique based on a RT-PCR with degenerate primers targeting a 287 bp fragment located on the conserved N terminus of the morbillivirus NP gene, followed by MseI RFLP, in order both to confirm the detection of the virus and to distinguish DMV from CDV. Both carnivores and cetaceans tissues (brain, lung and lymph node) presenting evidence of morbillivirus infection (MI) were analyzed. RT-PCR positive samples were typed by RFLP analysis and then sequenced to confirm the RFLP results. This method was applied during the last morbillivirus cetacean die-off occurred in the Mediterranean basin in 2013, when there was the urgent need of a rapid and economic method to investigate among causes of death on stranded cetaceans. This new technique has proved to be a valuable, reliable, simple and relatively inexpensive diagnostic tool easily applicable also in limited-resource laboratories.

Control of Mosquito-Borne Diseases in Northwestern Italy: Preparedness from One Season to the Next.

INTRODUCTION: Mosquito-borne diseases (MBDs) are spreading worldwide due to globalization and climate change, representing a threat for both humans and animals. Of great concern are the infections caused by viruses belonging to the Flavivirus genus as West Nile virus (WNV) and Usutu virus (USUV) transmitted by Culex sp. or Dengue virus and Zika virus (ZIKV), transmitted by Aedes sp. This work describes the surveillance protocol enforced in Piedmont (Northwestern Italy) to control MBDs spread, focusing on the activities performed on mosquitoes during the 2015 vector season. MATERIALS AND METHODS: From July to October, mosquitoes were fortnightly sampled in 50 selected sites according to risk factors with CDC dry ice-baited traps and BG-Sentinel traps baited with BG-Lure and dry ice. Adults were counted, identified to species level, pooled, and screened for flaviviruses using different reverse transcription-PCR protocols and sequencing. Finally, phylogenetic analysis was performed on a dataset including 2014 and 2015 WNV sequences and reference sequences retrieved from GenBank. RESULTS AND DISCUSSION: A total of 17,000 mosquitoes, grouped in 730 pools, were tested. Five pools of Culex pipiens were positive for WNV Lineage 2 in Novara, Alessandria, Vercelli, and Torino Provinces. One pool of C. pipiens and one pool of Anopheles maculipennis s.l. were positive for USUV in Vercelli and Alessandria Provinces. In Vercelli Province one pool of C. pipiens resulted positive both for WNV and USUV. Control measures were quickly implemented. Phylogenetic analyses showed that the WNV Lin 2 sequences from Piedmont region cluster with those circulating in Northeastern Italy in the previous years. Given the positive trend in WNV activity compared to 2014 and the emergence caused by other flavivirus as ZIKV, the level of attention for the 2016 vector season may be increased and this surveillance protocol could represent an important tool for public health authorities.

Dolphin Morbillivirus in a Cuvier's Beaked Whale (Ziphius cavirostris), Italy.

Dolphin morbillivirus (DMV) has caused several mortality events in Mediterranean striped (Stenella coeruleoalba) and bottlenose (Tursiops truncatus) dolphins populations since 19; in the last 5 years, the virus was reported to infect new hosts in this basin, such as fin whales (Balaenoptera physalus), sperm whales (Physeter macrocephalus), and even a harbor seal (Phoca vitulina). Very recently, a calf Cuvier's beaked whale (Ziphius cavirostris) calf stranded on the Southern Italian coastline with mild pathological findings suggestive of morbilliviral infection, received the first confirmation of DMV infection in this species by biomolecular evidences on lung tissue. This new cross-species infection report, along with 19% of the cetaceans specimens examined by the Italian Stranding Network being found positive to DMV, support the hypothesis of an endemic circulation of this virus among Mediterranean cetaceans.

Meningoencephalitis and Listeria monocytogenes, Toxoplasma gondii and Brucella spp. coinfection in a dolphin in Italy.

Listeria monocytogenes, Toxoplasma gondii and Brucella spp. can infect a wide range of species, including humans. In cetaceans, meningoencephalitis has been associated with T. gondii and Brucella spp. infection, whereas to our knowledge, L. monocytogenes infection has not previously been reported. Meningoencephalitis and L. monocytogenes, T. gondii and Brucella spp. were identified by means of both direct and indirect laboratory techniques in an adult female striped dolphin Stenella coeruleoalba found stranded in January 2015 on the Ligurian Sea coast, northwestern Italy. The animal was emaciated, and histopathology disclosed severe meningoencephalitis. The nature of the inflammatory response and intra-lesional protozoa were consistent with a mixed infection by L. monocytogenes, T. gondii and Brucella spp. We believe this is an unprecedented case of infection by 3 zoonotic pathogens and also the first bacteriologically confirmed case report of neurolisteriosis in cetaceans. Cerebral toxoplasmosis and neurobrucellosis may have led to the animal's disorientation and stranding, with L. monocytogenes having likely exacerbated the coinfection leading to the demise of this dolphin.

Detection of West Nile and Usutu Viruses in Italian Free Areas: Entomological Surveillance in Piemonte and Liguria Regions, 2014.

West Nile virus and Usutu virus have established in different parts of Italy over the past 10 years. Piemonte and Liguria Regions (Northwestern Italy) are known to be nonendemic areas, despite the presence of competent vectors and environmental conditions conducive to maintaining infection. This work evidences for the first time, through an entomological surveillance implemented on the basis of risk factor approach, the presence of West Nile and Usutu viruses in Piemonte and Liguria Regions (Northwestern Italy).