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Intentional poisoning represents a serious risk to domestic and wild animals, and it can be an environmental and human health issue as well . This paper is a retrospective study, which covers a decade, based on animal poisoning cases and poisoned baits that were submitted for diagnostic examinations to the Veterinary Medical Research Institute for Piedmont, Liguria and the Aosta Valley (IZS-PLVA) in Liguria region. All data were collected through a passive surveillance system introduced in Italy by a decree of the Ministry of Health in January 2009.43.2% of the animal poisoning cases were confirmed by toxicological analysis, whereas toxic agents were detected in 31.1% of the baits. The most affected animal species were dogs and cats, followed by synanthropic birds,. Only 4% of the total poisoning events analysed involved wild animals and cases of livestock poisoning were minimal. An increased number of cases in January, March, April and August was noticed, but no seasonal trend was detected. The most affected areas were the ones with the highest level of urbanization and population density. The major cause of the poisonings and the most common substances detected in the examined baits were anticoagulants whereas cholinesterase inhibitors, organochlorine pesticides and carbamates were detected in a minor number of cases.This study raises concerns about deliberate animal poisoning in ligurian region and highlights the necessity to fight this phenomenon as it endangers animals, humans and environment.

Brucella ceti infections have been increasingly reported in cetaceans. In this study, we analyzed all cases of B. ceti infection detected in striped dolphins stranded along the Italian coastline between 2012 and 2021 ( N = 24). We focused on the pathogenic role of B. ceti through detailed pathological studies, and ad hoc microbiological, biomolecular, and serological investigations, coupled with a comparative genomic analysis of the strains. Neurobrucellosis was observed in 20 animals. The primary histopathologic features included non-suppurative meningoencephalitis ( N = 9), meningitis ( N = 6), and meningoencephalomyelitis ( N = 5), which was also associated with typical lesions in other tissues ( N = 8). Co-infections were detected in more than half of the cases, mostly involving Cetacean Morbillivirus (CeMV). The 24 B. ceti isolates were assigned primarily to sequence type 26 (ST26) ( N = 21) and, in a few cases, ST49 ( N = 3). The multilocus sequence typing (cgMLST) based on whole genome sequencing (WGS) data showed that strains from Italy clustered into four genetically distinct clades. Plotting these clades onto a geographic map suggests a link between their phylogeny and the topographical distribution. These results support the role of B. ceti as a primary neurotropic pathogen for striped dolphins and highlight the utility of WGS data in understanding the evolution of this emerging pathogen.

Canine leishmaniosis (CanL) is an emerging zoonosis caused by Leishmania infantum and transmitted in southern Europe by phlebotomine sand flies of the subgenus Phlebotomus (Larroussius). Endemic foci of CanL have been recorded in northern continental Italy since early 1990s and attributed to the northward expansion of vector populations due to climatic changes in association with travelling/relocated infected dogs from the southern Mediterranean littoral. In this study, further spread of endemic Leishmania foci was monitored during 2018-2019 in five regions (Aosta Valley, Piedmont, Lombardy, Veneto and Friuli-Venezia Giulia), with focus to territories where investigations were not performed, or they have been inconclusive. Clinical cases of CanL identified by local veterinary practitioners and confirmed by reference diagnosis centers were regarded as autochthonous if their origin from, or travel to, areas endemic for CanL were excluded in the previous ≥2 years. Around these index cases, i) serosurveys for L infantum were carried out where indicated, ii) sampling from potential autochthonous cases in healthy or clinically-suspected resident dogs was intensified by collaborating veterinary practitioners, and iii) suitable sites were investigated for the presence of competent phlebotomine vectors. Fifty-seven municipalities whose enzootic status of CanL was unreported before 2018, were identified as endemic. The stability of 27 foci recorded over the past decade, was also confirmed. Competent phlebotomine vectors, mainly Phlebotomus perniciosus, were collected for the first time in 23 municipalities. The newly recorded endemic municipalities appear to be distributed over a west-to-east decreasing gradient: 30 in Piedmont, 21 in Lombardy, 4 in Veneto and 2 in Friuli-Venezia Giulia. As regards Veneto, it should be noted that a relatively restricted territory was investigated as several municipalities of the region had already been surveyed and detected as endemic for CanL in the past. Cold climate conditions of the easternmost region of Friuli-Venezia Giulia bordering non-endemic territories of Slovenia, are probably less favorable to L infantum transmission., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The genus Pennella comprises hematophagous parasites of marine aquatic species, including cephalopods, marine mammals, and pelagic fish. Nine species have been officially included in the genus Pennella plus another six species inquirendae . They are most often found in the host's musculature, without penetrating internal organs. For the present study, 83 hearts from swordfish ( Xiphias gladius ) caught in the Mediterranean Sea were sampled and immediately fixed in formalin for histopathological analysis. In total, 10 (12.05%) hearts were found to be parasitized by copepods of the genus Pennella . Macroscopically, there was mild-to-severe fibrinous pericarditis with atrial wall thickening and multiple parasitic nodules. Histologically, the parasitic nodules were surrounded by an inflammatory-necrotizing reaction. Parasitic infestation by Pennella spp. is common in pelagic fish and in swordfish, in particular. Here, however, we report atypical cardiac localization. A future area of focus is the evaluation of cardiac Pennella spp. infestation by histopathology and genetic identification of the parasites.

Intentional poisoning represents a serious risk to domestic and wild animals, and it can be an environmental and human health issue as well . This paper is a retrospective study, which covers a decade, based on animal poisoning cases and poisoned baits that were submitted for diagnostic examinations to the Veterinary Medical Research Institute for Piedmont, Liguria and the Aosta Valley (IZS-PLVA) in Liguria region. All data were collected through a passive surveillance system introduced in Italy by a decree of the Ministry of Health in January 2009. 43.2% of the animal poisoning cases were confirmed by toxicological analysis, whereas toxic agents were detected in 31.1% of the baits. The most affected animal species were dogs and cats, followed by synanthropic birds,. Only 4% of the total poisoning events analysed involved wild animals and cases of livestock poisoning were minimal. An increased number of cases in January, March, April and August was noticed, but no seasonal trend was detected. The most affected areas were the ones with the highest level of urbanization and population density. The major cause of the poisonings and the most common substances detected in the examined baits were anticoagulants whereas cholinesterase inhibitors, organochlorine pesticides and carbamates were detected in a minor number of cases. This study raises concerns about deliberate animal poisoning in ligurian region and highlights the necessity to fight this phenomenon as it endangers animals, humans and environment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Salmonella spp. is an important zoonotic agent. Wild boars might host this pathogen in the intestinal tract and might represent a risk for Salmonella spp. transmission to humans. Wild boars are widely spread in Liguria, due to the environmental characteristics of the region. The aim of the study was the isolation, typing, and investigation of antimicrobial susceptibility of the isolated strains of Salmonella spp. During the 2013-2017 hunting seasons, 4335 livers of wild boars were collected and analyzed for the presence of Salmonella spp. A total of 260 strains of Salmonella spp. were isolated and characterized, with a prevalence of 6%. The isolated strains belonged to all six Salmonella enterica subspecies. Most of them were identified as Salmonella enterica subs. enterica of which 31 different serotypes were identified. The dominating serotype identified was S. Enteritidis. The antimicrobial resistance profiles of the isolated strains were analyzed against sixteen molecules. Of the isolated strains, 94.6% were resistant to at least one of the tested antimicrobials. This study showed the circulation of resistant Salmonella spp. strains in the wild boar population living in this area of Italy, underling the potential risk for these animals to disseminate this pathogen and its antimicrobial resistances.

Yersinia enterocolitica ( Ye ) is a very important zoonosis andwild boars play a pivotal role in its transmission. In the last decade, the wild boar population has undergone a strong increase that haspushed them towards urbanized areas, facilitating the human-wildlife interface and the spread of infectious diseases from wildlife to domestic animals and humans. Therefore, it is important to know the serotype, antimicrobial resistance and presence of pathogenicity genes of Yersinia enterocolitica ( Ye ) isolated in species. From 2013 to 2018, we analyzed the liver of 4890 wild boars hunted in Liguria region; we isolated and serotyped 126 Ye positive samples. A decisive role in the pathogenicity is given by the presence of virulence genes; in Ye isolated we found ystB (~70%), ymoA (45.2%), ail (43.6%) and ystA (~20%). Moreover, we evaluated the susceptibility at various antimicrobic agents (Ampicillin, Chloramphenicol, Enrofloxacin, Gentamicin, Kanamycin, Trimethoprim-Sulfamethoxazole, Sulfisoxazole, Ceftiofur and Tetracycline). The antibiotic resistance was analyzed, and we found a time-dependent increase. It is important to shed light on the role of the wild boars as a reserve of potentially dangerous diseases for humans, and also on the antibiotic resistance that represents a public health problem.

Dolphin morbillivirus (DMV) is considered an emerging threat having caused several epidemics worldwide. Only few DMV genomes are publicly available. Here, we report the use of target enrichment directly from cetacean tissues to obtain novel DMV genome sequences, with sequence comparison and phylodynamic analysis. RNA from 15 tissue samples of cetaceans stranded along the Italian and French coasts (2008-2017) was purified and processed using custom probes (by bait hybridization) for target enrichment and sequenced on Illumina MiSeq. Data were mapped against the reference genome, and the novel sequences were aligned to the available genome sequences. The alignment was then used for phylogenetic and phylogeographic analysis using MrBayes and BEAST. We herein report that target enrichment by specific capture may be a successful strategy for whole-genome sequencing of DMV directly from field samples. By this strategy, 14 complete and one partially complete genomes were obtained, with reads mapping to the virus up to 98% and coverage up to 7800X. The phylogenetic tree well discriminated the Mediterranean and the NE-Atlantic strains, circulating in the Mediterranean Sea and causing two different epidemics (2008-2015 and 2014-2017, respectively), with a limited time overlap of the two strains, sharing a common ancestor approximately in 1998.

Toxoplasmosis represents an important public health issue, with the consumption of raw or undercooked meat being a major way of human infection. The role of beef in the transmission of the parasite to humans is questioned due to lower quantity of tissue cysts compared with other meat-producing species. However, the habit of consuming raw beef is regionally diffused, and the risk posed by Toxoplasma gondii infection in cattle should not be overlooked. Therefore, to update information on T. gondii in cattle reared in Italy, a multicentric seroepidemiological survey was designed and implemented in four Northern regions (Liguria, Lombardy, Piedmont, and Trentino Alto Adige) and Sardinia. Overall, a convenience sampling was performed, collecting 1444 serum samples from 57 beef cattle herds. Thirteen beef breeds were sampled, besides cross-breed; bovines age varied from 3 months to over 12 years. Sera were tested with a commercial ELISA for the detection of anti-T. gondii antibodies. Individual and herd data were analyzed by binary logistic regression analysis. A T. gondii seroprevalence of 10.2% was recorded, with differences among regions and values ranging from 5.3% in Liguria to 18.6% in the Piedmont region (p value = 0.0001). Both young and adult animals and males and females tested positive, without any significant difference (age and gender: p value > 0.05). Lower seroprevalence values were recorded in cattle born in Italy (8.7%) if compared with animals imported from abroad (13.4%) (p value = 0.046). The spread of T. gondii in beef cattle destined to Italian consumers is confirmed, suggesting the need of continuous monitoring of the infection.

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., Competing Interests: The authors have declared that no competing interests exist.

Leptospirosis is a re-emerging and widespread zoonosis, worldwide distributed, due to a wide variety of wild and domestic animal species able to act as natural or accidental hosts. During last years, in Europe, as in Italy, wild boar (Sus scrofa) population is increased. This animal represents a reservoir for different etiological agents, such as Leptospira. The aim of this investigation was to evaluate the prevalence of Leptospira spp. in wild boar hunted in Liguria region (Italy) during two-year hunting seasons. From 611 hunted wild boar, kidneys were collected. DNA was extracted from each organ and different targets were used to detect pathogenic (lipL32 gene), intermediate (16S rRNA gene) and saprophytic (23S rRNA gene) Leptospira by Taqman-based RealTime-PCR assays. Overall, kidneys were sampled from 282 adults, 155 sub-adults and 174 young wild boar (in total 314 males and 298 females). By RealTime PCR 77 kidneys were positive and, among these, 74 resulted positive for pathogenic (96.10%) and 3 (3.90%) for intermediate Leptospira. No significant differences in pathogenic Leptospira infection ratio were detected between male (11.50%) and female (12.75%). Only 13 sub-adult animals (8.39%) resulted infected by pathogenic Leptospira; 23 young animals (13.22%) and 38 adult animals (13.47%) were positive. The results of this study confirmed the importance of wild boar in the epidemiology of leptospirosis, which is able to infect other animal species (domestic and wild) including humans. Rarely, intermediate Leptospira could be able to infect wild boar with a renal localization that can contribute to their shedding and circulation., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Canine distemper (CD) may pose a serious threat to Alpine wild carnivores and affect their population dynamics. Since 2006, the strain Europe Wildlife 2006-09, a distinct CD virus subgroup within viral lineage Europe 1 (EU1) characterized by increased virulence and host range expansion, has been linked to multiple CD outbreaks in Alpine wild carnivores. The aim of this study was to fill knowledge gaps about ongoing Alpine outbreaks of CD. To do this, we report on the circulation of canine distemper virus (CDV) and outbreaks of CD in Alpine wild carnivores in northwest Italy. A specific diagnostic protocol applied to a sample of 548 wild carnivores collected between January 2013 and December 2015 revealed the circulation of CDV belonging to the EU1 lineage. All isolates were carriers of amino-acid mutations defining the cluster Europe Wildlife 2006-09. A self-maintained multihost pathogen system may have developed in northwest Italy in which interspecies transmission from red foxes ( Vulpes vulpes ) to other noncanid species enhanced pathogen maintenance in the system.

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.

Between 2015 and the beginning of 2018 (January-March), 30 cetaceans were found stranded along the Ligurian Sea coast of Italy. Necropsies were performed in 22 cases and infectious diseases resulted the most common cause of death. Three striped dolphins, showed a severe coinfection involving the monophasic variant of Salmonella Typhimurium (Salmonella 1,4,[5],12:i:-). The isolates were characterized based on antimicrobial resistance, Multiple-Locus Variable-number tandem-repeat Analysis (MLVA) and whole-genome sequencing (WGS). All isolates demonstrated the same multidrug resistant genotype (ASSuT isolates), showed three different MLVA profiles, two of which closely related, and were identified as Sequence Type 34. Moreover, Single nucleotide polymorphisms (SNP) analysis confirmed strong correlations between two out of the three isolates. To our knowledge, S. 1,4,[5],12:i:-, one of the most common serovars in cases of human infection and food sources worldwide, has not previously been described in marine mammals, and reports of Salmonella-associated disease in free-ranging cetaceans are rare. These results highlight the role of cetaceans as sentinel species for zoonotic and terrestrial pathogens in the marine environment, suggest a potential risk for cetaceans and public health along the North Western Italian coastline and indicate cetaceans as a novel potential reservoir for one of the most widespread Salmonella serovars.

We compared results of a serum immunofluorescence assay (IFA) and lymph node quantitative PCR (qPCR) in dogs classified as exposed, infected, or sick because of leishmaniasis. We also determined how IFA or qPCR results changed in response to treatment and reflected different clinical and clinicopathologic improvement of dogs. We included 108 dogs in our retrospective study: 12 exposed, 25 infected, and 71 sick, as classified according to Canine Leishmaniasis Working Group standards. Between-group comparison showed higher IFA values ( p < 0.01) for sick dogs; qPCR values were higher for sick than infected dogs ( p < 0.01). A novel clinical and clinicopathologic score was created and applied to 50 sick dogs. Using this score, 41 were reclassified as partially recovered (PR) within 3 mo, and 37 as totally recovered (TR) 3-6 mo after presentation. Statistically significant differences in IFA values were found between the sick and TR dogs ( p < 0.01), but not between sick and PR dogs ( p = 0.98). During follow-up, qPCR revealed a progressive decrease in parasite load, with a statistically significant difference in sick versus PR ( p < 0.01), sick versus TR ( p < 0.01), and PR versus TR ( p < 0.01) dogs. A decrease of 1 point in the clinical score corresponded to 1.3 Leishmania/μL qPCR decrease ( p < 0.01) and decrease of 1:42 in IFA ( p < 0.01). Our findings confirm that the clinical status of dogs affected by leishmaniasis is closely related to parasite load and antibody level, both before and after treatment.

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.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Visceral leishmaniasis (VL) is a zoonotic infectious disease with a severe impact on humans and animals. Infection is transmitted by phlebotomine sand flies, and several domestic and wild mammals act as reservoirs for the infection, so the prompt detection of infected hosts is crucial to preventing and controlling the spread of the disease and its transmission to humans. A rapid and portable tool for VL diagnosis based on the lateral flow immunoassay (LFIA) technology is described herein. The device exploits a highly specific chimeric recombinant antigen as the recognition element for capturing anti-leishmanial antibodies, and protein A labelled with gold nanoparticles as the signal reporter. The LFIA shows excellent diagnostic sensitivity (98.4%), specificity (98.9%), and agreement with serological reference methods for diagnosing canine VL. The long-term stability of the LFIA device was confirmed based on six months of storage at room temperature or 4 °C, and the qualitative response of the device was not affected by limited thermal stress. The use of the broadly specific protein A means that the LFIA can be readily adapted to diagnose VL in dogs (the main reservoir for human infection) and other mammals, thus further assisting efforts to control the spread of VL. Graphical abstract A rapid and portable diagnostic tool for visceral leishmaniasis (VL) based on lateral flow immunoassay (LFIA) technology. The presence of anti-leishmanial antibodies is revealed through the binding of these antibodies to a highly specific chimeric antigen. Employing a broadly specific signal reporter (protein A labelled with gold nanoparticles) enables the LFIA to be easily adapted to diagnose VL in different animals.

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.

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.

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.

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&#95;IZSPLV&#95;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&#95;IZSPLV&#95;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.

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.

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., (Copyright © 2017. Published by Elsevier B.V.)

In September 2014, seven sperm whales were stranded along Italy's Adriatic coastline. Postmortem investigations on 3 female adult whales and 1 male fetus carried by the largest female revealed molecular and immunohistochemical evidence of dolphin morbillivirus infection. A possible role of the virus in the stranding event was considered.

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).

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.

This paper reports the findings of a study on the presence of various species of filarial nematodes in dogs in Liguria, north-west Italy, a region traditionally considered free from the disease. Between 2009 and 2012 blood samples were taken from 365 dogs in rural areas in Liguria. The blood samples were then submitted to Knott's test, histochemical staining, polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) for Dirofilaria immitis antigens. Overall, 35 of the 365 dogs were positive using Knott's test for microfilariae (prevalence 9.6%; 95% confidence interval (CI): 6.6-12.6%). Acanthocheilonema reconditum was the most prevalent species (8.0%), while Dirofilaria repens (1.4%) and Dirofilaria immitis (0.6%) were less common. One co-infection by D. repens and A. reconditum was observed. All morphological identifications were confirmed by histochemical staining and PCR. In addition, a retrospective analysis of data on D. immitis antigens in 11,363 samples of canine sera was carried out. Sera were collected and analysed for D. immitis antigens by the Istituto Zooprofilattico Sperimentale (IZS) of Piedmont, Liguria and Aosta Valley (Imperia section) between 2004 and 2013 during annual tests for leishmaniasis on autochthonous dogs throughout Liguria. Serological data from IZS showed an overall seroprevalence of 0.65% (95% CI: 0.50-0.80%) for D. immitis throughout the region. The present study updates the epidemiological map of canine filarial infections in Italy and suggests the need for surveillance and prophylaxis in Liguria.

Meat products from HEV-infected reservoir animal species are capable of transmitting HEV to humans and represent a public health concern. Human HEV cases have been linked to the consumption of raw or undercooked pig liver sausages, pork, and game meats, such as wild boars and deer worldwide. Direct exposure to swine or wild game species might also represent a source of HEV transmission especially for veterinarians, hunters, or butchers. A limited amount of data is available on HEV prevalence in wild boars in Italy and no data are available for other wild game species intended for human consumption. In this study, the circulation of HEV in four different animal species hunted in north-western Italy was evaluated to gain insight into the infection levels and the genetic diversity of the virus in such animal populations. Liver samples of 372 wild boars, 30 roe deer, 47 European hares and 38 coypus were analyzed for HEV RNA by real-time RT-PCR; positive samples were then sequenced and submitted to phylogenetic analysis. HEV RNA was detected in the livers of 7/372 (1.9%) wild boars tested, while no sample was positive for roe deer, European hare, and coypu. Phylogenetic analysis showed that wild boar HEV sequences belonged to HEV subtypes 3e, 3c, and 3f. Our results indicate that HEV is circulating only in wild boar among the considered game species in north-western Italy and suggest a potential zoonotic risk related to handling and/or consumption of raw or undercooked meat and products made of the liver from this species.

This study reports the results of seroepidemiological investigations carried out against Morbillivirus, Toxoplasma gondii and Brucella spp. on blood serum samples collected from 70 cetacean specimens found stranded along the Italian coastline between 1998 and 2014. A total number of 23 serum samples (32.8%) obtained from Stenella coeruleoalba, Tursiops truncatus, Balaenoptera physalus and Globicephala melas harboured anti-Morbillivirus neutralizing antibodies. Ten sera (16%) collected from S. coeruleoalba and T. truncatus were found positive against T. gondii, while no antibodies against Brucella spp. were found. These data reveal that stranded cetaceans provide a unique opportunity for monitoring the health status of free-ranging animals living in the Mediterranean Sea, in order to investigate the level of exposure of cetacean populations to selected infectious agents representing a serious threat for aquatic mammals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Extraintestinal nematodes of the red fox (Vulpes vulpes) are a wide group of parasites that infect wild and domestic carnivores and occasionally humans. Nematodes in the cardiopulmonary system, stomach, urinary apparatus and muscle tissue of 165 red foxes (Vulpes vulpes) from north-west Italy (Liguria and Piedmont) were investigated between 2009 and 2012. Of the cardiopulmonary nematodes, a high prevalence of Angiostrongylus vasorum and Eucoleus aerophilus (syn. Capillaria aerophila) was found, 78.2% and 41.8% respectively; Crenosoma vulpis (15.8%) and Filaroides spp. (4.8%) were also found. Spirocerca lupi (23.5%), Aonchotheca putorii (syn. Capillaria putorii) (8.6%) and Physaloptera spp. (2.5%) were detected in the stomach and Pearsonema plica (syn. Capillaria plica) (56.8%) in the bladder. Eucoleus boehmi (syn. Capillaria boehmi) was also detected in the nasal cavities of one of the two foxes examined. A coprological examination revealed eggs of E. aerophilus, A. putorii, S. lupi, Physaloptera spp. and eggs of intestinal parasites. Filarial worms were absent in all the 165 animals examined, nor was there evidence of Trichinella spp. in any of the foxes. The foxes were found to host a high prevalence of many species of extraintestinal nematodes. The prevalence of A. vasorum in foxes found in the present study is among the highest in Europe. In addition, to the best of our knowledge, E. boehmi and Filaroides spp. have never been reported before in this host in Italy.

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) contribute to the dietary intake of total PCBs. They are consumed via ingestion of contaminated fish and fishery products. Thus, it is important to quantify their levels in aquatic organisms in order to evaluate the risks associated with fish consumption by humans. In 2013, an advisory against consumption of fish from the River Roya (IT) was disseminated after NDL PCBs were found to exceed maximum levels set by EU Regulations. We investigated the presence of NDL PCBs in eel, trout and barbel from the River Roya. We found concentrations in the range of 9.2-27.6 ng g(-)(1)ww in barbel, 9.2-97.0 ng g(-1)ww in trout and 9.0-239.5 ng g(-1)ww in eel. The distribution of congeners among different species did not show significant variations and was characterized by a higher proportion of 153, 138 and 180 congeners, according to their high resistance to metabolic degradation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Although rare in developed countries, most acquired human cases of hepatitis E virus (HEV) infection are associated with travel to developing countries where HEV is endemic. Increasingly, however, sporadic, non-travel-related HEV cases have been reported in developed countries. In Italy, only two studies to date have investigated the presence of HEV in wild boars. Here, we report a serological and virological survey of HEV in wild boar populations in northwestern Italy. During the hunting season, 594 serum and 320 liver samples were collected and screened for antibodies to HEV and HEV RNA. Overall, the seroprevalence was 4.9 %, and HEV RNA was detected in 12 liver samples (p = 3.7 %). No serum samples tested positive for HEV RNA. Phylogenetic analysis of the ORF2 region revealed that the isolates clustered within genotype 3, subtypes 3e and 3f, and were closely related to HEV strains previously detected in domestic pigs farmed in the same geographic area. Although the routes of viral transmission are still poorly understood, our data show that HEV genotypes 3e and 3f circulate in wild boars in northwestern Italy. Also, they provide evidence that autochthonous HEV infections in Italy could also be linked to wild boar populations, suggesting an increased risk for domestically acquired HEV infection in humans through wild animals. The HEV sequences determined in this study may be useful for comparing present and future human isolates to identify transmission events between wild boar, humans, and farmed pigs. Similarly to other more commonly known zoonotic agents, HEV should be included in national or regional disease surveillance programs for wild animals.