In vitro phenotypic characterisation of two genotype I African swine fever viruses with genomic deletion isolated from Sardinian wild boars.

African swine fever virus (ASFV) causes a devastating disease affecting domestic and wild pigs. ASF was first introduced in Sardinia in 1978 and until 2019 only genotype I isolates were identified. A remarkable genetic stability of Sardinian ASFV isolates was described, nevertheless in 2019 two wild boar isolates with a sustained genomic deletion (4342 base pairs) were identified (7303WB/19, 7212WB/19). In this study, we therefore performed in vitro experiments with monocyte-derived macrophages (moMФ) to unravel the phenotypic characteristics of these deleted viruses. Both 7303WB/19 and 7212WB/19 presented a lower growth kinetic in moMФ compared to virulent Sardinian 26544/OG10, using either a high (1) or a low (0.01) multiplicity of infection (MOI). In addition, flow cytometric analysis showed that both 7303WB/19 and 7212WB/19 presented lower intracellular levels of both early and late ASFV proteins. We subsequently investigated whether deleted virus variants were previously circulating in wild boars in Sardinia. In the four years preceding the last genotype I isolation (February 2015-January 2019), other eight wild boar isolates were collected, all belonging to p72 genotype I, B602L subgroup X, but none of them presented a sustained genomic deletion. Overall, we observed the deleted virus isolates in Sardinia only in 2019, at the end of a strong eradication campaign, and our data suggest that it might possess an attenuated phenotype in vivo. A better understanding of ASFV evolution in endemic territories might contribute to development of effective control measures against ASF.

Genotype I hepatitis A virus introduction in Italy: Bayesian phylogenetic analysis to date different epidemics.

Despite a significant decrease in acute hepatitis A in the last 2 decades in Italy, outbreaks were observed occurring mostly in southern Italy. In this study, Bayesian phylogenetic analysis was used to analyze the origin of these epidemics. With this aim, 5 different data sets of hepatitis A virus sequences were built to perform genotyping by the neighbor-joining method to estimate the evolutionary rates by using a Bayesian Markov chain Monte Carlo approach and to investigate the demographic history by independent Markov chain Monte Carlo runs enforcing both a strict and relaxed clock. The estimated mean value of the evolutionary rate, representing Ia and Ib strains, was 1.21 × 10-3 and 2.0 × 10-3 substitutions/site/year, respectively. The Bayesian maximum clade credibility tree of hepatitis A virus (HAV) Ia and Ib strains showed that Italian sequences mostly formed separate clusters. The root of the time for the most recent common ancestor (tMRCA) for HAV Ia and Ib strains dated back to 1981 and to 1988, respectively, showing in both cases different epidemic entrances. Phylodynamic analysis showed that genotype Ia increased in 1997, when the Apulia epidemic started, then suffered a bottleneck, probably consequent to vaccination and to the herd immunity, followed by a new increase in virus population in the years 2013-2014 consequent to the epidemic caused by the ingestion of mixed frozen berries. A similar trend without an evident bottleneck was observed also in the case of genotype Ib. In conclusion, the Bayesian phylogenetic analysis represents a good tool to measure the effectiveness of the public health plans used for HAV control.

Trend of Pathogenic Vibrio parahaemolyticus Occurrences in Bivalve Molluscs Harvested in Sardinian Coastal Environments Between 2011 and 2018.

The aim of the present study was to evaluate Vibrio parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2013 and 2015. The prevalence of pathogenic V. parahaemolyticus isolates is based on the detection of the two major virulence genes thermostable direct hemolysin (tdh) and thermolabile hemolysin (trh) To assess changes between 2011 and 2018 in the prevalence of V. parahaemolyticus in bivalve molluscs, we compared our results with those of previous investigations. In total, 2,933 samples were collected: 1,079 in 2013, 1,288 in 2014, and 566 in 2015. The mean prevalence of V. parahaemolyticus in shellfish was 3.5% in 2013, 1.7% in 2014, and 3.5% in 2015. The highest percentage of positive samples in 2013 and 2014 was observed in clams (3.5% and 2.7%, respectively), whereas in 2015, it was reported in oysters (15.1%). By comparing the sampling period of 2011-2014 with that of 2015-2018, an increase in the prevalence of V. parahaemolyticus was observed in shellfish (p < 0.05). In parallel, 208 potentially enteropathogenic V. parahaemolyticus strains were identified through the years 2011-2018 and, in particular, 10 trh+ and six tdh+ isolates. Our present study provides information regarding trends of V. parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2011 and 2018 suggesting that the prevalence varies depending on the sampling period and shellfish species.

Influence of seasonality on the presence of okadaic acid associated with Dinophysis species: A four-year study in Sardinia (Italy).

In Sardinia (Italy), bivalve molluscs production plays an important role in the trade balance. Diarrhoetic shellfish poisoning (DSP), an intoxication caused by the ingestion of bivalve molluscs that have accumulated high levels of Okadaic acid (OA), may represent a serious risk for the public health and a remarkable economic loss for the producers. Aim of this work was to improve knowledge about the repeatability of OA accumulation phenomena in various seasons trying to understand whether or not there was a trend. Also, the interaction between toxic algae and OA accumulation was examined. In this study, data of lipophilic toxins, water temperature and abundance of DSP-producing microalgal species were collected in a four-year period (2015-2018) in coastal production areas of Sardinia. Several episodes of OA positive values (>160 eq µgAO/Kg pe, Reg 853/04) were recorded during the study period in different production areas of Sardinia and in different seasons. A seasonal repeatability of OA accumulation in molluscs was observed in some production areas; moreover, different temporal gaps between the presence of toxic algae and OA accumulation were reported. Toxicity was observed almost exclusively in Mytilus galloprovincialis Lamark (99%), being this matrix the most abundant species bred in Sardinia.

The Bayesian reconstruction and the evolutionary history of Salivirus type 1 and type 2: the worldwide spreading.

INTRODUCTION: Salivirus (SalV) represents an emerging problem in public health especially during the recent years. In this study, the Bayesian evolutionary history and the spread of the virus through the different countries have been reported. METHODOLOGY: a database of 81 sequences of SalV structural VP1 fragment were downloaded from GenBank, aligned and manually edited by Bioedit Software. ModelTest v. 3.7 software was used to estimate the simplest evolutionary model fitting the sequence dataset. A Maximum-Likelihood tree has been generated using MEGA-X to test the "clockliness" signal using TempEst 1.5.1. The Bayesian phylogenetic tree was built by BEAST. Homology modelling was performed by SWISS-Model and protein variability evaluated by ConSurf server. RESULTS: the phylogenetic tree showed a clade of SalV A2 and three main clades of SalV A1, revealing several infections in humans in South Korea, India, Tunisia, China, Nigeria, Ethiopia and USA. The Bayesian maximum clade credibility tree and the time of the most common recent ancestor dated back the root of the tree to the year 1788 with the probable origin in USA. Selective pressure analysis revealed two positive selection sites, His at 100th and Leu at 116th positions that at the homology modelling resulted important to guarantee protein stability and variability. This could contribute to the development of new mutations modifying the clinical features of this evolving virus. CONCLUSIONS: Bayesian phylogenetic and phylodynamic represented a useful tool to follow the transmission dynamic of SalV and to prevent new epidemics worldwide.

New evidence of pectenotoxins in farmed bivalve molluscs from Sardinia (Italy).

Several planktonic dinoflagellates can produce lipophilic phycotoxins that represent a significant threat to public health as well as to shellfish and fish farming. Poisoning related to some of these toxins is categorised as diarrhetic shellfish poisoning. We analysed 975 shellfish samples from Tortoli in the central-eastern region of Sardinia (Italy) from January 2016 to March 2020, to investigate the prevalence of different lipophilic marine biotoxins in mollusc bivalves. The results highlighted the predominant presence of toxins belonging to the okadaic acid group in all samples with toxin concentrations exceeding legal limits, and revealed the new occurrence of pectenotoxins in oysters and clams with a winter seasonality in recent years. The origin of shellfish toxicity was associated with the same Dinophysis species, mainly D. acuminata. Based on both these results and other precedents, monitoring and recording systems are strongly recommended.

Maternal multiple sclerosis is not a risk factor for neurodevelopmental disorders in offspring.

BACKGROUND: Childhood neurodevelopmental disorders (NDDs), including specific learning disorders (SLD), attention deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD), are pathogenically linked to familial autoimmunity and maternal immune-mediated diseases during pregnancy. OBJECTIVE: We studied maternal MS as a potential risk factor for NDDs occurrence in offspring. METHODS: MS and control mothers were subjected to questionnaires to ascertain NDD diagnosis in their progeny and the occurrence of both autoimmune and neurodevelopment disorders in their families. Suspected NDD cases were evaluated to confirm or rule out the diagnosis. RESULTS: Of the 322 MS women, 206 (64%) have 361 children; of these, 27 (7.5%) were diagnosed with NDD (11% ADHD; 22% ASD; 67% SLD). NDD-risk in offspring was associated to family history of autoimmunity and to NDDs both in MS and non-MS mother families (r = 0.75; p = 0.005) whereas it was not associated to maternal MS. CONCLUSIONS: For the first time, we demonstrate that maternal MS does not predispose children to higher risk for NDD. On a mechanistic view, we suggest that the intrinsic organ-specific nature of MS does not impair the mother-child cross-talk in decidua nor does it influence fetal neurodevelopment.

Presence, Seasonal Distribution, and Biomolecular Characterization of Vibrio parahaemolyticus and Vibrio vulnificus in Shellfish Harvested and Marketed in Sardinia (Italy) between 2017 and 2018.

ABSTRACT: In the present study, we investigated the presence, seasonal distribution, and biomolecular characteristics of Vibrio parahaemolyticus and Vibrio vulnificus in samples of bivalve mollusks (Mytilus galloprovincialis, Crassostrea gigas, and Ruditapes decussatus) harvested and marketed in Sardinia (Italy) between 2017 and 2018. A total of 435 samples were submitted for qualitative determination of Vibrio spp., V. parahaemolyticus, and V. vulnificus. Potentially enteropathogenic isolates were detected with biomolecular methods. The overall prevalence of Vibrio spp. was 7.6%. The highest Vibrio prevalence was found in R. decussatus (8.3%). The prevalences of V. parahaemolyticus and V. vulnificus were 2.7 and 4.8%, respectively. Higher prevalences of V. parahaemolyticus and V. vulnificus were found in R. decussatus (4.2%) and C. gigas (6.2%), respectively. Only two pathogenic V. parahaemolyticus strains were recovered (genotypes: tdh- and trh+; tdh+ and trh-), both from M. galloprovincialis. None of the isolates were tdh+ and trh+. Pathogenic Vibrio infections are often underestimated, and human infections are increasing in Europe. European data on the true distribution of Vibrionaceae are scarce, and the results of the present study highlight the need of constant monitoring to update the distribution of pathogenic vibrios.

Malaria in an asylum seeker paediatric liver transplant recipient: diagnostic challenges for migrant population.

Transplanted patients are particularly exposed to a major risk of infectious diseases due to prolonged immunosuppressive treatment. Over the last decade, the growing migration flows and the transplant tourism have led to increasing infections caused by geographically restricted organisms. Malaria is an unusual event in organ transplant recipients than can be acquired primarily or reactivation following immunosuppression, by transfusion of blood products or through the transplanted organ. We report a rare case of Plasmodium falciparum infection in a liver transplanted two years-old African boy who presented to one Italian Asylum Seeker Center on May 2019. We outlined hereby diagnostic challenges, possible aetiologies of post-transplantation malaria and finally we summarized potential drug interactions between immunosuppressive agents and antimalarials. This report aims to increase the attention to newly arrived migrants, carefully evaluating patients coming from tropical areas and taking into consideration also rare tropical infections not endemic in final destination countries.

Outbreak of acute hepatitis A associated with men who have sex with men (MSM) in North Sardinia 2017-2018.

INTRODUCTION: We describe an outbreak of hepatitis A among men who have sex with men (MSM) which evolved in Sassari (Italy), between January 2017 and December 2018, close to a contextual of large concurrent hepatitis A outbreak reported in Europe and recently, in Italy. METHODOLOGY: HAV RNA detection and molecular characterization was performed from serum samples and/or stool by RT - PCR of VP1/2A junction region that ranges from nt. 2,873 to nt. 3,376. The phylogenetic correlation of the circulating hepatitis A strains was assessed by sequencing method according to the HAVNET protocol. RESULTS: 10 Acute Hepatitis A virus (AHA)-positive cases, 8 of which were among men who have sex with men (MSM) were identified. All patients were tested at the time of hospitalization for the presence of anti-HIV antibodies, only two MSM resulted co-infected by HIV. No differences were observed in median age (37 years vs. 41 years, P-value = 0.14), severity or duration of hospitalization between seropositive and HIV-negative men. Phylogenetic analysis was conducted in 2 cases and revealed two distinct sequences of genotype IA linking to clusters recognized in MSM in other European countries in 2016. CONCLUSION: Our study reported a recent increase of notified hepatitis A cases attributable to cases in the European interconnectedness of MSM in Sassari, a small locality. Hepatitis A vaccination for MSM in Sardinia region is recommended; however, our data emphasize the need of hepatitis A screening and vaccination not only for MSM with occasional partners but also for those in monogamous relationships.

Migrants rescued on the Mediterranean Sea route: nutritional, psychological status and infectious disease control.

INTRODUCTION: North Africa has become a key migratory hub where a large number of migrants attempt the journey by sea from the Libyan coastline to the south of Europe. In this humanitarian disaster scenario, the Mediterranean route has been one of the most used by illegal boats. METHODOLOGY: In this report, the state of physical and psychological health of a cluster of Eritrean migrants, escaped from Libya and rescued in the Mediterranean Sea after a shipwreck, was described by epidemiological, clinical and laboratory investigations. RESULTS: Data suggest that despite the majority  of the migrants being apparently in good health upon a syndromic surveillance approach, most of them suffered a decline in psychological status as well as severe malnutrition. The emergence of infectious diseases, related to poor living conditions during the journey, is not a rare event. CONCLUSION: The present report highlights the risks of failures of the syndromic medical approach in the setting of the extremely challenging migration route and underlines migrant frailties consequent to a prolonged journey and long period of detention. These stressors, which can degrade the initial health condition of traveling migrants, can lead to a premature "exhausted migrant effect" that should be carefully investigated in order to avoid the early emergence of diseases related to frailty.

Vaccination campaign strategies in recently arrived migrants: experience of an Italian reception centre.

INTRODUCTION: Control of vaccine preventable diseases, while constituting a priority of European health policies, is challenged by migrations from countries with suboptimal levels of immunization coverage. We report here two different types of vaccination campaign strategy in one of the bigger Italian asylum seekers' centres. The vaccination service staff of the local national health institute came monthly during the first three years of observation, while in the last year, the vaccinations were offered directly upon arrival of migrants in the asylum seekers' centre. METHODOLOGY: we performed a descriptive cross-sectional study that analysed data collected from the database of the internal healthcare facility and ARVA Target tool, regarding vaccinations performed from 2013 to 2017 in the asylum seekers' centre. RESULTS: In the four years of observation period the asylum seekers centre hosted 3941 migrants. Among them, 85% were vaccinated during their stay, for a total of 4252 vaccinations administered, covering 95% of minors and 85% of adults. During the study period, there was an important increase from an average of 10.5% of migrants vaccinated in the first three years to 66% in the last year, when vaccines were delivered directly upon arrival in the centre. CONCLUSIONS: To improve the rate of immunization in migrants, the first requirement is a strong collaboration with the local vaccine services and the second,vaccinations must be carried out when migrants arrive at the asylum seekers' centre, avoiding any delay.

Bacterial and Viral Investigations Combined with Determination of Phytoplankton and Algal Biotoxins in Mussels and Water from a Mediterranean Coastal Lagoon (Sardinia, Italy).

Calich Lagoon is a Mediterranean coastal lagoon located along the northwestern coast of Sardinia (Italy). The connection to marine and fresh water determines the high productivity of this coastal lagoon. Despite its great potential and the presence of natural beds of bivalve mollusks (Mytilus galloprovincialis), the lagoon has not yet been classified for shellfish production. In this study, through a multidisciplinary approach, the presence of several bacterial pathogens (Escherichia coli, Salmonella spp., and Vibrio spp.) and viral pathogens (hepatitis A virus and norovirus genogroups I and II) was evaluated from March 2017 to February 2018. In addition, phytoplankton composition in lagoon waters and associated algal biotoxins (paralytic and diarrhetic shellfish poisoning) in mussels were also monitored. The aim of this study was to provide useful data to improve knowledge about their seasonal presence and to assess the potential risk for public health, as well as to provide input for future conservation and management strategies. In mussels, Salmonella spp. were found in spring, along with E. coli, but Salmonella spp. were not found in autumn or winter, even though E. coli was detected in these seasons. Vibrio parahaemolyticus was found in autumn and winter, but not in spring. Norovirus genogroups I and II were found in winter samples. None of the bacteria were found in summer. Algal biotoxins have never been detected in mussel samples. Among potentially harmful phytoplankton, only Pseudo-nitzschia spp. were present, mainly in summer. The results showed that a possible bacterial and viral contamination, together with the presence of potentially toxic microalgae, is a real problem. Therefore, the development of natural resource management strategies is necessary to ensure the good quality of waters and guarantee the protection of consumers.

Detection of Dinophysis Species and Associated Okadaic Acid in Farmed Shellfish: a Two-year Study from The Western Mediterranean Area.

INTRODUCTION: Diarrhoetic shellfish poisoning (DSP), an alimentary intoxication known to lead to intestinal symptoms, and caused by toxins produced by some dinoflagellates (including several Dinophysis), represents a serious threat to public health. The aim of this paper was to provide information about the occurrence and abundance of potentially toxic harmful algal species causing DSP, and the associated concentration of okadaic acid (OA) toxins. The departing assumption was that in the study area there was an increase in the presence both of Dinophysis species and OA and its derivates that could result in a risk to the health of seafood consumers. MATERIAL AND METHODS: During 2015-2016, water and shellfish samples were collected in the Mediterranean area (Sardinia, Italy). Dinophysis cells were counted according to Utermöhl's method from water samples, while mass spectrometry was used to identify lipophilic toxins in molluscs. RESULTS: A total of 46 non-compliant samples of Mytilus galloprovincialis were observed. Their non-compliance concerned their OA levels above the legal limit. Among toxic dinoflagellates, D. acuminata and D. sacculus were the species found mostly during DSP events. CONCLUSION: No cases of human intoxication have been reported, but continuous surveillance of toxic phytoplankton is necessary to predict and prevent its harmful effects on human health.

Yessotoxin detection in bivalve molluscs: A case study from coastal mussel farms (Sardinia, Italy).

This work reports the first communication relating to the presence of yessotoxins in Mytilus galloprovincialis from coastal mussel farms (Sardinia, western Mediterranean) detected during 2008 and 2013 through a monitoring programme. The paper emphasizes how the changes both in yessotoxin permitted limits and used methods, established by legislation, have influenced the interpretation of the obtained results. Consequently, the samples that resulted negative during 2008 would have been positive until August 2013 and negative from September 2013 up to now, and the samples that were positive in 2013 would have been positive in 2008 and negative nowadays, according to Regulation currently in force. Regular monitoring of biotoxins demonstrated that, although yessotoxins have been rarely present in the past in Sardinia, they may cause toxicity in shellfish. So, it's important to keep up on legislation's changing and laboratory methods.

Preliminary Study on Norovirus, Hepatitis A Virus, Escherichia coli and their Potential Seasonality in Shellfish from Different Growing and Harvesting Areas in Sardinia Region.

Edible lamellibranch molluscs can be involved in foodborne disease and infections of varying severity. They are filter feeding animals able to retain and concentrate in their organism bacteria, parasites, viruses and biotoxins marine algae present in their external environment. Major shellfish harvesting and relaying areas from different areas in Sardinia region were defined and studied by analysing different physicochemical parameters in the water and the levels of Escherichia coli (E. coli), Norovirus (NoVs) genogroup I (NoVGI), NoVs genogroup II (NoVGII) and hepatitis A virus (HAV) in the shellfish harvested and farmed from 2009 to 2011. During that period the identification of the viral agents was carried out by one step real-time reverse transcriptase-polymerase chain reaction and Escherichia coli according to ISO TS 16649-3:2005 standard method. A total of 1266 shellfish samples were tested for NoVGI, NoVGII, HAV and faecal indicators. Norovirus contamination was found in 337 samples (26.6%); only one sample of mussels was positive for HAV (0.08%); while E. coli prevalence was 3.8% in shellfish. The probability of observing shellfish samples positive for NoVs, HAV and E. coli presence was associated with harvesting, growing and relaying areas, period of sampling, environmental parameters, animal species (P<0.05). Although the higher prevalence rate of human enteropathogenic viruses was found in the winter period, we did not observe a significant relationship between the effect of seawater temperature (seasonality) and NoVs presence all over the study period; in fact, according to statistical analysis, the presence of human enteric viruses does not appear to be related to water temperature.