Draft genome of three isolates of Listeria monocytogenes isolated from Stenella coeruleoalba in Italy.

Listeria monocytogenes is the etiological agent of the listeriosis. Here, we described three draft genome sequences of L. monocytogenes isolated in Italy from stranded individuals of the striped dolphin Stenella coeruleoalba. All the genomes have been molecular typed through the multilocus sequence typing to identify the phylogenetic lineage, clonal complex, sublineage, and serogroup.

An extremely rare serovar of Salmonella enterica (Yopougon) discovered in a Western Whip Snake (Hierophis viridiflavus) from Montecristo Island, Italy: case report and review.

Reptiles, including snakes, can be asymptomatically infected with multiple pathogen microorganisms, including Salmonella spp., which is considered an important concern for public and animal health. Small and uninhabited isles are quite ecologically different from mainland and represent interesting fields of study, to discover unexpected biological and microbiological aspects of their wild inhabitants. This work reports the presence of the very rare Salmonella enterica serovar Yopougon, isolated in a carcass of a native wild snake (Hierophis viridiflavus) from an Italian uninhabited island of Mediterranean Sea, Montecristo. To our knowledge, S. enterica serovar Yopougon was previously isolated only once 34 years earlier in Ivory Coast, from a human fecal sample. In the present study, we present the genomic characterization of the new isolate, the phylogenetic comparison with the previously isolated S. enterica serovar Yopougon strain of human origin and with other sequences available in public databases. In addition, an extensive review of available data in the literature and from our case history is provided. Our finding represents an example of the ability of some pathogens to travel for very long distances within their hosts and then to infect others, even from different taxa.

Isolation and characterization of Fusarium spp. From unhatched eggs of Caretta caretta in Tuscany (Italy).

Sea Turtle Egg Fusariosis (STEF) is a worldwide emergent fungal disease affecting eggs and causing embryos mortality in turtle's nests such as those of Caretta caretta. It is caused by a complex of species belonging to Fusarium genus, particularly those included in the Fusarium Solani Species Complex (FSSC). During the samplings carried out in summer 2020 along the Tuscany coastlines (Italy), C. caretta eggs showed clinical signs resembling those caused by STEF. A total of 32 fungal isolates were obtained from lesioned eggs whose molecular characterization allowing identifying as belonging to FSSC / Neocosmospora spp., Fusarium oxysporum Species Complex (FOSC) / F. oxysporum and Fusarium nodosum, i.e., fungal genera and speciesincluding also well-known plant pathogens. Isolates inoculated on several plant hosts did not result in any pathogenic activity but F. nodosum causing, on wheat spikes, disease symptoms.This is the first time F. nodosum has been isolated from portions of eggs showing evident signs of fungal infection. This work represents the first report of Fusarium spp. isolated from C. caretta eggs showing lesions resembling those caused by STEF on Tuscan coast thus posing a significant concern to loggerhead sea turtle conservation also in this region.

Brucella ceti Infection in Striped Dolphins from Italian Seas: Associated Lesions and Epidemiological Data.

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.

A Familiar Outbreak of Monophasic Salmonella serovar Typhimurium (ST34) Involving Three Dogs and Their Owner's Children.

Salmonella is a Gram-negative enteric bacterium responsible for the foodborne and waterborne disease salmonellosis, which is the second most reported bacterial zoonosis in humans. Many animals are potential sources of salmonellosis, including dogs, cats, and other pets. We report the case of an outbreak of salmonellosis in a family in central Italy, affecting two children and involving their three dogs as carriers. One of the children needed medical care and hospitalisation. Isolation and analysis of stool samples from the sibling and the animals present in the house were carried out. Serotyping allowed the identification of S. enterica subsp. enterica serovar Typhimurium in its monophasic variant for all the isolates. The results of whole-genome sequencing confirmed that the strains were tightly related. The minimum inhibitory concentration (MIC) test documented the resistance to ampicillin, sulfamethoxazole, and tetracycline. The origin of the zoonotic outbreak could not be assessed; however, the case study showed a clear passage of the pathogen between the human and non-human members of the family. The possibility of a transmission from a dog to a human suggests the need for further studies on the potential ways of transmission of salmonellosis through standard and alternative feed.

Potential SARS-CoV-2 Susceptibility of Cetaceans Stranded along the Italian Coastline.

Due to marine mammals' demonstrated susceptibility to SARS-CoV-2, based upon the homology level of their angiotensin-converting enzyme 2 (ACE2) viral receptor with the human one, alongside the global SARS-CoV-2 occurrence and fecal contamination of the river and marine ecosystems, SARS-CoV-2 infection may be plausibly expected to occur also in cetaceans, with special emphasis on inshore species like bottlenose dolphins (Tursiops truncatus). Moreover, based on immune and inflammatory responses to SARS-CoV-2 infection in humans, macrophages could also play an important role in antiviral defense mechanisms. In order to provide a more in-depth insight into SARS-CoV-2 susceptibility in marine mammals, we evaluated the presence of SARS-CoV-2 and the expression of ACE2 and the pan-macrophage marker CD68. Aliquots of tissue samples, belonging to cetaceans stranded along the Italian coastline during 2020-2021, were collected for SARS-CoV-2 analysis by real-time PCR (RT-PCRT) (N = 43) and Immunohistochemistry (IHC) (N = 59); thirty-two aliquots of pulmonary tissue sample (N = 17 Tursiops truncatus, N = 15 Stenella coeruleoalba) available at the Mediterranean Marine Mammal Tissue Bank (MMMTB) of the University of Padua (Legnaro, Padua, Italy) were analyzed to investigate ACE2 expression by IHC. In addition, ACE2 and CD68 were also investigated by Double-Labeling Immunofluorescence (IF) Confocal Laser Microscopy. No SARS-CoV-2 positivity was found in samples analyzed for the survey while ACE2 protein was detected in the lower respiratory tract albeit heterogeneously for age, gender/sex, and species, suggesting that ACE2 expression can vary between different lung regions and among individuals. Finally, double IF analysis showed elevated colocalization of ACE2 and CD68 in macrophages only when an evident inflammatory reaction was present, such as in human SARS-CoV-2 infection.

Parasites, Bacteria, and Associated Pathological Changes in the Digestive System of Diurnal and Nocturnal Raptors in Central Italy.

The knowledge of raptor pathogens and associated lesions may be extremely important to enhancing raptor conservation efforts and reducing pathogen spillover to humans and domesticated animals and vice versa. Parasite infections of the digestive system and associated bacteria and pathological changes were evaluated in deceased diurnal and nocturnal raptors in central Italy. Overall, the prevalence of parasites (nematodes, cestodes, trematodes, acanthocephalans, and protozoa) identified in the examined birds was 72.41%, and most of the positive raptors (71.42%) showed multiple parasite infections. Among bacteria, Salmonella typhimurium, Salmonella enterica subspecies diarizonae, Escherichia coli, Clostridium perfringens, Yersinia enterocolitica, and Pasteurella multocida were identified. The results obtained showed that both parasites and bacteria may cause severe lesions in the digestive system of diurnal and nocturnal raptors; parasites and bacteria may concur in causing these lesions; most severe lesions are caused by the interaction of multiple pathogens, both parasites and bacteria; and the same pathogen taxa are frequently associated with the same pathological changes. This study is the first report of S. typhimurium and S. enterica subspecies diarizonae in Buteo buteo, while Andracantha mergi, Spirocerca spp., Sarcocystis dispersa, Sarcocystis columbae, and Eumonospora spp. were recorded for the first time in Italy.

First Report on the Finding of Listeria mnocytogenes ST121 Strain in a Dolphin Brain.

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes the foodborne illness, listeriosis. Clonal complexes (CC), such as CC121, are overrepresented in the food production industry, and are rarely reported in animals and the environment. Working within a European-wide project, we investigated the routes by which strains are transmitted from environments and animals to food and the food production environment (FPE). In this context, we report, for the first time, the occurrence of a ST121 (CC121) strain isolated from a dolphin brain. The genome was compared with the genomes of 376 CC121 strains. Genomic comparisons showed that 16 strains isolated from food were the closest to the dolphin strain. Like most of the food strains analyzed here, the dolphin strain included genomic features (transposon Tn6188, plasmid pLM6179), both described as being associated with the strain's adaptation to the FPE. Like all 376 strains, the dolphin strain contained a truncated actA gene and inlA gene, both described as being associated with attenuated virulence. Despite this fact, the strain was able to cross blood-brain barrier in immunosuppressed dolphin exposed polychlorinated biphenyl and invaded by parasites. Our data suggest that the dolphin was infected by a food-related strain released into the Mediterranean Sea.

Dolphins Stranded along the Tuscan Coastline (Central Italy) of the "Pelagos Sanctuary": A Parasitological Investigation.

Parasite monitoring is considered a necessary step for cetacean management and conservation. Between February 2013 and July 2015, 26 dolphins (15 Stenella coeruleoalba, 10 Tursiops truncatus, and one Grampus griseus) stranded along the Tuscan coastline of the protected marine area "Pelagos Sanctuary", were examined. Organs, tissues, and faecal and blood samples taken from all animals were analysed by parasitological, immunological, and molecular techniques. Twenty-one out of 26 dolphins (80.77%) tested positive for at least one parasite species, and 13/15 (86.7%) S. coeruleoalba, 7/10 (70%) T. truncatus, and the single G. griseus were found positive. Identified parasites included the nematodes Skrjabinalius guevarai (7.69%, 2/26), Halocercus lagenorhynchi (3.85%, 1/26), Halocercus delphini (7.69%, 2/26), Stenurus ovatus (7.69%, 2/26), Crassicauda spp. (7.69%, 2/26); the trematodes Pholeter gastrophilus (26.92%, 7/26), Campula palliata (3.85%, 1/26); the cestodes Phyllobothrium delphini (42.31%, 11/26), Monorygma grimaldii (23.08%, 6/26), Tetrabothrium forsteri (7.69%, 2/26), Strobilocephalus triangularis (7.69%, 2/26), and the acanthocephalan Bolbosoma vasculosum (7.69%, 2/26). Moreover, 6/26 (23%) animals scored positive to Toxoplasma gondii at serology, but PCR confirmed the infection (T. gondii Type II genotype) in a single animal. In examined dolphins, obtained results showed a high prevalence of endoparasites, which included species considered as a cause of severe debilitation or death.

Epidemiologic Survey on Toxoplasma gondii and Trichinella pseudospiralis Infection in Corvids from Central Italy.

Free-ranging corvids-678 magpies (Pica pica) and 120 hooded crows (Corvus cornix) from nine protected areas of the Pisa province (central Italy)-were examined for Toxoplasma gondii and Trichinella pseudospiralis. The intracardiac blood clots from 651 magpies and 120 hooded crows were serologically examined for T. gondii. The DNA extracted from the hearts of seropositive birds was then used to perform a nested PCR for the amplification of the T. gondii B1 gene and for genotyping for SAG genetic markers. Breast muscle samples from 678 magpies and 91 hooded crows were tested by an artificial digestion method for Trichinella. Data were statistically analyzed. Forty-five (5.8%-41 magpies and four hooded crows) out of the 771 examined animals scored seropositive for T. gondii, with titers ranging from 1:25 to 1:100. T. gondii DNA was detected in 15 of the 45 positive birds and T. gondii genotypes II and III were identified. No positivity for T. pseudospiralis was found. No significant differences between the two species of corvids and among the different areas of origin were observed for seropositivity to T. gondii. This is the first extensive study on both T. gondii and T. pseudospiralis in magpies and hooded crows, as well as the first detection of T. gondii SAG genotypes in magpies.

Cetacean Poxvirus in Two Striped Dolphins (Stenella coeruleoalba) Stranded on the Tyrrhenian Coast of Italy: Histopathological, Ultrastructural, Biomolecular, and Ecotoxicological Findings.

Tattoo skin disease (TSD) is a poxviral disease typical of cetaceans. Two juvenile and well-preserved male striped dolphins (Stenella coeruleoalba), found stranded along the Tuscany and Latium coasts of Italy in 2015 and 2016, respectively, showed typical skin lesions ascribable to TSD. Histological, ultrastructural and biomolecular investigations confirmed a poxviral aetiology for the aforementioned skin lesions. To our knowledge, this should be the first report of TSD in cetaceans stranded along the Italian coastline. As organochlorines like PCBs and DDTs are known to be highly immunotoxic, the tissue loads of these contaminants were evaluated, in order to increase our knowledge on their potential role as well as on the relationships between the level of exposure to these pollutants and poxviral infection's occurrence.

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.

Leishmania infantum, Dirofilaria spp. and other endoparasite infections in kennel dogs in central Italy.

Prevalence and risk factors of Leishmania infantum, Dirofilaria spp. and other potentially zoonotic or canine-specific endoparasite infections were assessed in 639 kennel dogs from central Italy. To this end, individual blood and fecal samples were examined using parasitological, immunological and molecular techniques. The presence of compatible clinical pictures, as well as age and gender were considered as putative risks factors. To evaluate risk factors, multivariable analysis with logistic regression and univariable analysis with a Chi square test and a Fischer's exact test were performed. Overall, 52.6% of dogs (95% CI 48.6-56.5) were found positive, while 39.6% of dogs (95% CI 35.8-43.5) were infected by potentially zoonotic species. Leishmania infantum and Dirofilaria repens showed prevalences of 2.5% (95% CI 1.5-4.1) and 2.8% (95% CI 1.7-4.5), respectively. The prevalence of cardiorespiratory parasites was 7.8% (95% CI 5.9-10.3) and included the species Angiostrongylus vasorum, Eucoleus aerophilus, Eucoleus boehmi and D. immitis; the latter showed a prevalence of 0.2% (95% CI 0.001-1). Intestinal parasites were significantly prevalent (38.8%, 95% CI 35-42.7) and they consisted mainly of species of major zoonotic concern, including ancylostomatids, Toxocara canis, Giardia duodenalis, Dipylidium caninum, Taeniidae, Strongyloides stercoralis and Cryptosporidium parvum. Endoparasites were significantly prevalent in clinically suspected dogs. Leishmania infantum and cardiorespiratory nematodes were prevalent in older dogs, while intestinal parasites were prevalent in younger dogs. Results show high dog and public health risks in kennels in central Italy, and suggest the need for more effective control measures.

A New Multilocus Sequence Typing Scheme and Its Application for the Characterization of Photobacterium damselae subsp. damselae Associated with Mortality in Cetaceans.

Photobacterium damselae subsp. damselae (PDD) is a known pathogen of fish, humans and marine mammals. In this study, a Multilocus Sequence Typing (MLST) scheme based on six housekeeping genes (glp, gyrB, metG, pnt, pyrC, and toxR) was developed to better understand the PDD population structure and used to type 73 PDD isolates from cetaceans, mainly striped dolphins (Stenella coeruleoalba) involved in mortality episodes, and from a few marine chelonians. Five reference ATCC strains were also included in the study. Typing allowed the discrimination of groups of PDD strains isolated from different host species, at different times and from different geographic areas, suggesting that a clonal PDD group may have spread in the Tyrrhenian sea at the time of an Unusual Mortality Event (UME) among cetaceans, mainly striped dolphins, occurred in early 2013 along the Italian western coasts.

Mediterranean Fin Whales (Balaenoptera physalus) Threatened by Dolphin MorbilliVirus.

During 2011-2013, dolphin morbillivirus was molecularly identified in 4 stranded fin whales from the Mediterranean Sea. Nucleoprotein, phosphoprotein, and hemagglutinin gene sequences of the identified strain were highly homologous with those of a morbillivirus that caused a 2006-2007 epidemic in the Mediterranean. Dolphin morbillivirus represents a serious threat for fin whales.

Retrospective seroepidemiological investigations against Morbillivirus, Toxoplasma gondii and Brucella spp. in cetaceans stranded along the Italian coastline (1998-2014).

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.

The presence of Brucella ceti ST26 in a striped dolphin (Stenella coeruleoalba) with meningoencephalitis from the Mediterranean Sea.

Brucella spp. was isolated from brain, lung and intestinal lymph nodes of a dead adult male striped dolphin (Stenella coeruleoalba) found stranded on the Tyrrhenian coast (Tuscany, Italy) of the Mediterranean Sea in February 2012. Brucella spp. was associated with moderate to severe lesions of meningoencephalitis. A co-infection by Toxoplasma gondii was also demonstrated at brain level by means of molecular and histopathologic methods. The Brucella isolate was further characterized based on a fragment-specific polymerase chain reaction (PCR) approach, consisting of a set of five specific PCRs, targeting specific chromosomal IS711 locations for marine mammal Brucellae, as described previously. The isolate was thus classified as Brucella ceti I; V fragment-positive (or B. ceti dolphin type), according to previous studies. Multi Locus Sequence Analysis demonstrated that the isolate belongs to Sequence Type 26, while omp2 (omp2a and omp2b genes) sequence analysis further confirmed the isolate belonged to this group of strains. This is the first report of Brucella spp. from marine mammals in the Mediterranean Sea, and represents a further observation that this strain group is associated with hosts of the Family Delphinidae, and particularly with the striped dolphins, also in the Mediterranean area, thus constituting a further biological hazard of concern for this vulnerable subpopulation.