Development and validation of species-specific molecular diagnostic tool for Opisthorchis felineus (Digenea, Opisthorchiidae) metacercariae

Opisthorchis felineus (family Opisthorchiidae) is a parasitic flatworm representing a serious threat to humans in some countries. Opisthorchiasis occurs after consumption of raw or undercooked cyprinid fish infected by the metacercarial stage of the parasite. Due to its small size, detection of the parasite in fish fillet is time-consuming and difficult. Furthermore, isolated metacercariae can be identified to genus but not to species level using morphological features and molecular techniques are necessary. In this work, we describe the development of primers for a diagnostic PCR amplification of a 254-bp fragment of the cytochrome c oxidase I in the mitochondrion of Opisthorchis felineus metacercariae isolated from fish fillet, together with a validation protocol for this method

Study on the Effect of Relaying on Norovirus Reduction from Crassostrea gigas Oysters

Norovirus (NoV) is the most important cause of seafood-borne gastroenteritis worldwide, mainly associated with the consumption of raw oysters. NoV is often present in oysters that comply with existing control standards for shellfish. Therefore, the improvement of post-harvest treatments and practices can represent one of the main strategies to reduce the incidence of viral diseases related to shellfish. This study aimed to investigate long-term relays for the reduction of NoV levels in live oysters, during the high-risk cold months, by transferring the oysters from a more contaminated site to two sites with lower NoV levels. The efficacy of relaying was evaluated by analyzing oyster samples collected at days 0 (T0) and 30 (T30) for NoV levels using a real-time quantitative PCR (RT-qPCR). The NoV level at the relay sites was consistently lower than at the harvest site. The relay process for 30 days in seawater with a lower NoV level resulted in a decrease in the NoV load compared to day 0 with significant reductions depending on the site and genogroup of NoV considered. These results suggest that long-term relaying of oysters to reduce NoV levels is promising and could help growers to improve oyster safety; however, further investigations are needed

Effects of Essential Oils and Hydrolates on the Infectivity of Murine Norovirus

peer reviewedThe use of natural substances with antiviral properties might reduce foodborne viral diseases. In this study, we evaluated the virucidal effect of Citrus limon and Thymus serpyllum essential oils (EOs) and of Citrus Limon, Thymus serpyllum and Thymus vulgaris hydrolates on murine norovirus (MNV), a human norovirus surrogate. To assess the virucidal effect of these natural substances, the reduction in viral infectivity was estimated by comparing the TCID50/mL of untreated viral suspension and the viral suspension treated with hydrolates and EOs at different concentrations. The results showed a natural loss of infectivity of the untreated virus after 24 h of approx. 1 log. The EO (1%) of T. serpyllum, and hydrolates (1% and 2%) of T. serpyllum and T. vulgaris immediately caused a reduction in MNV infectivity of about 2 log but did not provide a further significant decrease after 24 h. Instead, the EO (1%) and hydrolate (1% and 2%) of C. limon exerted an immediate reduction in the viral infectivity of about 1.3 log and 1 log, respectively, followed by a further reduction in infectivity of 1 log after 24 h for the hydrolate. These results will allow for the implementation of a depuration treatment based on the use of these natural compounds

Hepatitis E Virus (HEV): Identification of Subtypes 3b and 3m in Wild Boar Population in Liguria Region, Italy

The wild boar is an important natural reservoir for the zoonotic transmission of the hepatitis E virus (HEV) around the world. In particular, HEV genotypes 3 and 4 are an emerging problem in industrialized countries, as the number of wild boars has increased, and their territory is ever closer to farms and populated areas. This study describes the HEV prevalence and geographic circulation among wild boar populations in the Ligurian region (Italy) during the period 2019–2022. Liver samples from 849 wild boars were analyzed for HEV RNA using real-time RT-PCR; positive samples were then subjected to sequencing and phylogenetic analysis. Overall, 6.7% of the wild boars were positive for HEV RNA; however, in the last two years, the percentage of positive animals almost doubled. Phylogenetic analysis showed that wild boar HEV sequences belonged to genotype 3 and clustered within subtypes 3a and 3c, and, for the first time in Italy, subtypes 3b and 3m were identified. Interestingly, 13 sequences could not be assigned to a recognized subtype. Furthermore, the results showed different circulations of identified subtypes across the territory. These findings increase the knowledge of HEV-3 heterogeneity in Italy and describe the role of wild boars in maintaining an active viral circulation in the environment

Norovirus Persistence in Oysters to Prolonged Commercial Purification

Depuration is generally the main treatment employed for bivalve mollusks harvested from contaminated sites. Commercial depuration has demonstrated to be effective for removal of bacterial pathogens, although it probably provides only limited efficacy against human enteric viruses. We evaluated the quantitative reduction of norovirus (NoV) genogroups I and II in naturally contaminated oysters after 1, 4, and 9 days of depuration. The process was conducted in an authorized depuration plant, and NoV concentration was determined by RT-qPCR according to ISO 15216-1:2017 method. Regardless of the NoV genogroup, our results showed no significant reduction in NoV concentration after 1 day of depuration. Higher mean reduction (68%) was obtained after 4 days of treatment, while no further increase was observed after 9 days. Overall, reduction was highly variable, and none of the trials showed statistically significant reduction in NoV RNA concentration at the end of each depuration period. Indeed, NoV concentration remained high in 70% of samples even after 9 days of depuration, with values ranging between 4.0 × 102 and 2.3 × 104 g.c./g. These results indicate that an extension of commercial depuration time does not appear to be effective for reducing or eliminating NoV in oysters

An Extensive investigation into the prevalence and the genetic and serological diversity of toxigenic <i>Vibrio parahaemolyticus</i> in Italian marine coastal waters

The relationship between Vibrio parahaemolyticus strains isolated from the aquatic environment and those isolated from cases of infection in humans is poorly understood due to the low prevalence of tdh- and/or trh-positive strains in the environment. To address this concern, it would be useful to analyse the genetic relationships among environmental and food strains and with reference to clinical isolates, also applying molecular typing methods. The aim of this study was to evaluate the prevalence of toxigenic V. parahaemolyticus in Italian coastal waters and seafood, to examine intra-species variability and to identify, using serotyping and pulsed-field gel electrophoresis (PFGE), relationships among strains from different sources, geographical origin and period of isolation. Of the 192 V. parahaemolyticus strains isolated in different Italian areas and examined in this study, 25 (13.0%) proved to carry the trh gene while none of the strains proved positive to the search by PCR for tdh and Group-Specific-toxRS genes. The prevalence of toxigenic strains in the Tyrrhenian Sea was significantly lower than that calculated for the Ligurian coasts. Regarding the sources of isolation, the higher prevalence of trh-positive V. parahaemolyticus was revealed in fish, followed by clams, plankton, oysters, mussels and lastly seawater. Within the toxigenic strains, 16 serotypes and 20 distinct PFGE patterns were identified. Two clusters, which included a total of 8 V. parahaemolyticus strains, were specifically associated with the North Adriatic Sea area and were stable over time. Our results demonstrate that trh-positive V. parahaemolyticus strains circulated in Italy in the period 2002–2009 with a prevalence higher than that reported from other European and extra-European countries, confirming that toxigenic V. parahaemolyticus is an emerging public health concern in Italy, regardless of its pandemic potential

Exploring the Astrovirome of Shellfish Matrices Using Nanopore Sequencing

Simple Summary Astroviruses are important human pathogens, associated with gastro-enteric disease in children and recently with encephalitis in immunocompromised patients. Astroviruses have also been identified in mammals, birds, lower vertebrates and invertebrates in association with enteric and extra-intestinal diseases or, in some cases, as components of the enteric virome without a clear link with specific clinical signs. As a proof of concept, we conjugated the versatility and broad reactivity of a commonly used consensus primer set, able to amplify in a nested RT-PCR protocol the RNA-dependent RNA polymerase (RdRp) of most members of the Astroviridae family, with a nanopore sequencing platform, in order to assess the potential of this approach to generate astroviromic data in complex matrices. Amplicons generated from mussels were used to generate libraries, either alone or in pools, and subjected to deep sequencing. Overall, we identified a variety of known and unknown RdRp sequence types, in most cases distantly related to astrovirus sequences available in the databases. Avian-origin astrovirus sequences were predominant, likely due to contamination of surface water. Astroviruses are important human enteric pathogens transmissible with contaminated food and water. Astroviruses have also been identified in mammals, birds, lower vertebrates and invertebrates. The genetic diversity of human and animal astroviruses poses a challenge for diagnostics and taxonomy. As a proof of concept, we used a panastrovirus consensus primer set, able to amplify in a nested RT-PCR protocol a 400-nt-long fragment of the RNA-dependent RNA polymerase of most members of the Astroviridae family, in conjunction with a nanopore sequencing platform, to generate information on the astrovirome in filter-feeding mollusks. Amplicons generated from bivalve samples were used to generate libraries for deep sequencing. In three samples, only one unique RdRp sequence type was obtained. However, in seven samples and in three barcodes with eleven pooled samples, we identified a variety of known and unknown RdRp sequence types, in most cases distantly related to astrovirus sequences available in the databases. In total, 37 different sequence contigs were generated. Avian-origin astrovirus sequences were predominant, likely due to contamination of shellfish harvesting waters by marine birds. Astroviruses of the aquatic eco-system were also identified, whereas human astroviruses were not detected

In Vivo Evaluation of the Potential of Thyme and Lemon Hydrolates as Processing Aids to Reduce Norovirus Concentration during Oyster Depuration

peer reviewedIn this study, we evaluated the use of hydrolates, co-products of essential oil distillation, as processing aids to improve the depuration process of Pacific oysters (Crassostrea gigas) as a postharvest method aimed at reducing the norovirus (NoV) viral load. Live oysters were kept in water to which hydrolates of Thymus serpyllum and Citrus limon at 1% were added for 24 h. The concentration of NoV was quantified using the ISO 15216-1 quantitative real-time RT-PCR method in the oyster digestive tissue both before and after the treatment. The results showed a significant reduction of 0.2 log in the NoV GII concentration after 24 h of treatment with 1% C. limon hydrolate. Conversely, treatment with T. serpyllum did not appear to reduce the concentration of NoV compared to the control. Additionally, a sensory analysis was conducted through a blind survey comparing untreated and treated oysters. No changes in the sensory and physical characteristics of the oysters were observed, except for a decrease in the marine flavour intensity, which was positively perceived by consumers. These results indicate that the addition of hydrolates of C. limon at 1% during depuration might represent a promising processing aid for enhancing both the safety and acceptability of live oysters

Assessing the health status of farmed mussels (Mytilus galloprovincialis) through histological, microbiological and biomarker analyses

The Gulf of La Spezia (northern Tyrrhenian Sea, Italy) is a commercially important area both as a shipping port and for mussel farming. Recently, there has been increased concern over environmental disturbances caused by anthropogenic activities such as ship traffic and dredging and the effects they have on the health of farmed mussels. This paper reports the results of microbiological and histological analyses, as well as of measurement of several biomarkers which were performed to assess the health status of mussels (Mytilus galloprovincialis) from four rearing sites in the Gulf of La Spezia. Mussels were collected between October 2015 and September 2016 and histological analyses (including gonadal maturation stage), as well as the presence of pathogenic bacteria (Vibrio splendidus clade, V. aestuarianus and V. harveyi), viruses (Herpes virus and ostreid Herpes virus 1) and protozoa (Marteilia spp., in the summer season only) were carried out on a monthly basis. Conversely, biomarker responses in haemocyte/haemolymph (total haemocyte count, haemocyte diameter and volume, lysozyme and lactate dehydrogenase activities in cell-free haemolymph, and micronuclei frequency) and in gills and digestive gland (cortisol-like steroids and lipid peroxidation levels), were evaluated bimonthly. Microbiological data indicated that mussels contain a reservoir of potentially pathogenic bacteria, viruses and protozoa that in certain environmental conditions may cause a weakening of the immune system of animals leading to mortality episodes. The percentage of parasites detected in the mussels was generally low (9.6% for Steinhausia mytilovum, that is 17 samples out of 177 examined females; 3.4% for Proctoeces maculatus; 0.9% for Mytilicola intestinalis and 2% for ciliated protozoa), while symbiont loads were higher (31% for Eugymnanthea inquilina and Urastoma cyprinae). Interestingly, a previously undescribed haplosporidian was detected in a single mussel sample (0.2%) and was confirmed by in situ hybridization. Cells morphologically similar to Perkinsus sp. trophozoites were observed in 0.7% of the mussels analysed; however, infection with Perkinsus spp. could neither be confirmed by ISH nor by PCR. Different pathological aspects, such as host defence responses and regressive/progressive changes were detected in the gills, digestive glands, gonads and mantle. Only one single case of disseminated neoplasia (0.2%) was observed. As for the biomarker evaluation, the MANOVA analysis revealed the statistically significant effect that the variable \u201csampling site\u201d had on the biological parameter measured, thus suggesting that the multibiomarker approach was able to differentiate the rearing sites. \ua9 2018 Elsevier Inc