Genomic Characterization of Listeria monocytogenes and Other Listeria Species Isolated from Sea Turtles.

Listeria monocytogenes is a ubiquitous pathogen found both in the environment and food. It can cause listeriosis in a wide range of animals as well as in humans. Investigations on presence, spread and virulence are still limited to terrestrial and human environments. Embracing the One Health Approach, investigating the presence and spread of L. monocytogenes in marine ecosystems and among wildlife, would provide us with useful information for human health. This study investigated the presence of L. monocytogenes and Listeria spp. in two species of sea turtles common in the Mediterranean Sea (Caretta caretta and Chelonia mydas). A total of one hundred and sixty-four carcasses of sea turtles (C. caretta n = 161 and C. mydas n = 3) stranded along the Abruzzo, Molise, Campania, and Calabria coasts, were collected. Brain and fecal samples were taken, enriched, and cultured for the detection of Listeria spp. From the specimens collected, strains of L. monocytogenes (brain n = 1, brain and feces n = 1, multiorgan n = 1 and feces n = 1), L. innocua (feces n = 1 and brain n = 1), and L. ivanovii (brain n = 1) were isolated. Typical colonies were isolated for Whole Genome Sequencing (WGS). Virulence genes, disinfectants/metal resistance, and antimicrobial resistance were also investigated. L. monocytogenes, L. innocua, and L. ivanovii were detected in C. caretta, whilst only L. monocytogenes and L. innocua in C. mydas. Notable among the results is the lack of significant differences in gene distribution between human and sea turtle strains. Furthermore, potentially pathogenic strains of L. monocytogenes were found in sea turtles.

Exposure of Mytilus galloprovincialis to Microplastics: Accumulation, Depuration and Evaluation of the Expression Levels of a Selection of Molecular Biomarkers.

Microplastic contamination is a growing marine environmental issue with possible consequences for seafood safety. Filter feeders are the target species for microplastic (MPs) pollution because they filter large quantities of seawater to feed. In the present study, an experimental contamination of Mytilus galloprovincialis was conducted using a mixture of the main types of MPs usually present in the seawater column (53% filaments, 30% fragments, 3% granules) in order to test the purification process as a potential method for removing these contaminants from bivalves intended for human consumption. A set of molecular biomarkers was also evaluated in order to detect any variations in the expression levels of some genes associated with biotransformation and detoxification, DNA repair, cellular response, and the immune system. Our results demonstrate that: (a) the purification process can significantly reduce MP contamination in M. galloprovincialis; (b) a differential expression level has been observed between mussels tested and in particular most of the differences were found in the gills, thus defining it as the target organ for the use of these biomarkers. Therefore, this study further suggests the potential use of molecular biomarkers as an innovative method, encouraging their use in next-generation marine monitoring programs.

Species of mosquitoes present in Abruzzo and Molise and their possible role as vector of Usutu and West Nile viruses.

In 2019, entomological survey on mosquitoes was carried out in Abruzzo and Molise regions in central Italy to obtain data on local mosquito fauna. Collection sites were selected based on a previous ecoregion classification of the territory.  From 2019 to 2021 virological surveillance for West Nile virus (WNV) and Usutu virus (USUV) on mosquitoes was carried out in the same regions, selecting ecoregions where virus circulation and vector presence were more likely,  all mosquitoes were collected and identified, and the female mosquitoes were sorted in 3046 pools and tested for the presence of WNV and USUV by Real-time PCR. All pools tested negative for WND, while USUV was detected in 7 pools of Aedes caspius collected in Molise region, 17 pools of Culex pipiens s.l. (2 collected in Molise, 15 in Abruzzo), and 1 pool of Culiseta longiareolata collected in Molise. These results suggests the presence of an USUV enzootic cycle, maintained by Culex pipiens s.l. and Aedes caspius in both Italian regions, as well as providing a useful picture in terms of species presence and abundance for both regions. Ecoregions proved to be a very valuable tool in determining high risk areas for vector borne diseases.

Vector Competence of Italian Populations of Culicoides for Some Bluetongue Virus Strains Responsible for Recent Northern African and European Outbreaks.

The distribution of Bluetongue virus (BTV) in Europe can be represented by two distinct and interconnected epidemiological systems (episystems), each characterized by different ecological characteristics and vector species. This study investigated the vector competence of Italian populations of Culicoides imicola and Culicoides obsoletus/scoticus to some representative BTV strains after artificial oral infection. The BTV strains were selected according to their ability to spread to one or both episystems and included BTV-4 ITA, responsible of the recent Italian and French BTV-4 outbreaks; the BTV-2 strain which caused the first BTV incursion in Italy, Corsica, and Balearic Islands; BTV-4 MOR, responsible for the epidemic in Morocco; and BTV-8, the strain which spread through Europe between 2006 and 2008. Blood-soaked cotton pledgets and Hemotek membrane feeder using Parafilm® membrane were used to artificially feed midges. For each population/strain, recovery rates (positive/tested heads) were evaluated using serogroup- and serotype-specific RT-PCR. The trial demonstrated that, except for the Abruzzo population of C. obsoletus/C. scoticus, which was refractory to BTV-4 MOR infection, all the investigated Culicoides populations are susceptible to the selected BTV strains and that, if prompt vaccination programs and restriction measures had not been implemented, BTV-2 and BTV-4 MOR could have spread all over Europe.

Isolation and genome sequences of two Feline Morbillivirus genotype 1 strains from Italy.

Feline morbillivirus (FeMV) is a novel viral paramyxovirus detected in cats. FeMV is suspected to be associated to tubulointerstitial nephritis, but its pathogenic role is far to be clearly understood.  In this short communication, we report the whole genome coding sequences of the first two FeMV strains isolated in Italy.

Diagnosis and characterization of canine distemper virus through sequencing by MinION nanopore technology.

Prompt identification of the causative pathogen of an infectious disease is essential for the choice of treatment or preventive measures. In this perspective, nucleic acids purified from the brain tissue of a dog succumbed after severe neurological signs were processed with the MinION (Oxford Nanopore Technologies, Oxford UK) sequencing technology. Canine distemper virus (CDV) sequence reads were detected. Subsequently, a specific molecular test and immunohistochemistry were used to confirm the presence of CDV RNA and antigen, respectively, in tissues. This study supports the use of the NGS in veterinary clinical practice with potential advantages in terms of rapidity and broad-range of molecular diagnosis.

Outbreak of porcine epidemic diarrhoea virus (PEDV) in Abruzzi region, central-Italy.

Here we report and characterize a porcine epidemic diarrhea (PED) outbreak which occurred in a swine fattening farm in the province of Teramo, Abruzzi region (central Italy), in January 2016. PED virus (PEDV) identification was determined by real-time RT-PCR performed on RNAs purified from fecal samples collected from two symptomatic pigs. Whole genome sequence (PEDV 1842/2016) was also obtained by next generation sequencing straight from RNA purified from one fecal sample. Genome comparison with extant global PEDV strains revealed a high nucleotide identity with recently reported European and American S-INDEL PEDVs. Efficient sequencing, share of genomic data combined with the implementation of epidemiological tools would be the ideal approach for study and analysis of transboundary infectious diseases as PED.

Analysis of bluetongue serotype 3 spread in Tunisia and discovery of a novel strain related to the bluetongue virus isolated from a commercial sheep pox vaccine.

Bluetongue (BT), is one of the OIE-listed major diseases of ruminants. Following the official report of BT virus serotype 3 (BTV-3) in a sheep in Cap Bon (Tunisia), blood and serum samples of ruminants were collected from some areas of Tunisia to further investigate the presence of this virus in the country. A quantitative real time RT-PCR has been first developed for the detection and quantitation of BTV-3 RNA from field specimens. Out of 62 collected blood samples, 23 were shown to be positive for BTV-3 RNA. Isolation on cell cultures was also possible from six samples. Genome sequencing revealed the circulation of two unrelated western strains of BTV-3, one circulating in Cap Bon and neighboring areas, and the other circulating nearby the border with Libya. The presence of a putative novel BTV serotype (BTV-Y TUN2017) in sheep introduced from Libya to Tunisia, genomically related to the BTV strain contaminating a commercially-available sheep pox vaccine and to BTV-26, has been also demonstrated. This finding highlights the pressing need for a prompt production and release of a novel inactivated BTV-3 vaccine to be used in case of emergence or proactively in the areas of Southern Europe at major risk of BTV introduction. The assessment of a novel vaccine will certainly exalt the role and importance of surveillance activities and collaboration with Northern African countries.

Competitive enzyme-linked immunosorbent assay using baculovirus-expressed VP7 for detection of epizootic haemorrhagic disease virus (EHDV) antibodies.

Epizootic haemorrhagic disease (EHD) is a vector-borne infectious viral disease of domestic and wild ruminants. EHD could spread from infected northern African countries in free territories like the EU; therefore, the availability of diagnostic assays would represent key components for adequate surveillance and control programs. In this study, the gene encoding the VP7 protein of EHD virus (EHDV) was expressed into a baculovirus-infected insect cell system. With this unpurified protein we developed a home-made competitive ELISA (cELISA) and a total number of 275 serum samples, originating from domestic and wild ruminants, were tested. 74/275 were previously shown to be positive for EHDV antibodies by a commercially available ELISA kit. A "very good" agreement was demonstrated when compared to a commercial ELISA kit (Cohen's kappa value=0.832). Samples which caused disagreement between the two assays originated from wildlife which highlights the need for further validation by using serum samples from wild animals.

Genome Sequence of Canine Adenovirus Type 1 Isolated from a Wolf (Canis lupus) in Southern Italy.

Canine adenovirus type 1 (CAdV-1), a DNA virus of the family Adenoviridae, causes infectious canine hepatitis, a highly contagious disease primarily affecting canids. In this report, we describe the isolation and whole-genome sequence of a CAdV-1 isolate from the liver of a free-ranging wolf (Canis lupus).

A newly developed BVDV-1 RT-qPCR Taqman assay based on Italian isolates: evaluation as a diagnostic tool.

A single-step TaqMan® RT-qPCR was developed for the detection of bovine viral diarrhea virus type 1 (BVDV-1), an important pathogen of cattle worldwide. The assay was based on conserved 5'UTR sequences of Italian BVDV-1 isolates. In order to establish a diagnostic protocol which simplifies sample collection and processing, the assay was tested on a variety of biological specimens collected from persistently infected calves. The samples analyzed included PBMCs, plasma, dry blood, ear notch and hair bulb. Time and costs required for the analysis of each type of specimen were compared. The RT-qPCR, whose lower limit of detection was 100 copies of viral RNA (1 TCID50), correctly identified all PI animals, irrespective of the type of specimen. The highest copy numbers were obtained from the RNAs extracted from PBMCs, ear notches and hair bulbs. Hair bulb-supernatants directly used as a template allowed identification of all PI animals. In conclusion, based on time and cost evaluation, the most effective and efficient protocol was the one based on the direct analysis of hair bulb-supernatants, avoiding the RNA extraction step.

Whole-Genome Sequence of a Suid Herpesvirus-1 Strain Isolated from the Brain of a Hunting Dog in Italy.

Suid herpesvirus-1 (SHV-1), a DNA virus of the family Herpesviridae, causes a severe and fatal disease in a wide range of mammals. Here, we report the whole-genome sequence of an SHV-1 isolated in Italy in 2014 from the brain of a hunting dog that suffered from an acute and severe disease.

Effect of 1,3-1,6 ß-Glucan on Natural and Experimental Deformed Wing Virus Infection in Newly Emerged Honeybees (Apis mellifera ligustica).

The Western Honeybee is a key pollinator for natural as well as agricultural ecosystems. In the last decade massive honeybee colony losses have been observed worldwide, the result of a complex syndrome triggered by multiple stress factors, with the RNA virus Deformed Wing Virus (DWV) and the mite Varroa destructor playing crucial roles. The mite supports replication of DWV to high titers, which exert an immunosuppressive action and correlate with the onset of the disease. The aim of this study was to investigate the effect of 1,3-1,6 ß-glucan, a natural innate immune system modulator, on honeybee response to low-titer natural and high-titer experimental DWV infection. As the effects exerted by ß-glucans can be remarkably different, depending on the target organism and the dose administered, two parallel experiments were performed, where 1,3-1,6 ß-glucan at a concentration of 0.5% and 2% respectively, was added to the diet of three cohorts of newly emerged honeybees, which were sampled from a Varroa-free apiary and harboured a low endogenous DWV viral titer. Each cohort was subjected to one of the following experimental treatments: no injection, injection of a high-copy number DWV suspension into the haemocel (experimental DWV infection) or injection of PBS into the haemocoel (physical injury). Control bees fed a ß-glucan-free diet were subjected to the same treatments. Viral load, survival rate, haemocyte populations and phenoloxidase activity of each experimental group were measured and compared. The results indicated that oral administration of 0.5% ß-glucan to naturally infected honeybees was associated with a significantly decrease of the number of infected bees and viral load they carried, and with a significant increase of the survival rate, suggesting that this natural immune modulator molecule might contribute to increase honeybee resistance to viral infection.