Cross-sectional study of hepatitis E virus (HEV) circulation in Italian pig farms.

Foodborne transmission is considered the main way of spreading zoonotic hepatitis E virus (HEV) infection in Europe. In recent years, the human cases of hepatitis E in subjects without history of travel in endemic areas have raised, suggesting that domestic HEV transmission is increasing. Pork products with or without liver, are often indicated as the source of many human foodborne HEV cases as well as small outbreaks. Pigs are recognized as the main reservoir of the zoonotic HEV-3 genotype, the most frequently detected in human cases in the EU. In the absence of a harmonized surveillance of HEV circulation, data on prevalence are heterogeneous but confirm a widespread circulation of HEV-3 in pig herds across EU. HEV-3 can pass through the food chain from farm to fork when infected animals are slaughtered. In Italy, several studies reported the circulation of HEV-3 in pig farms, but results are heterogeneous due to different methodologies applied. In the present study, we performed a survey over 51 pig herds belonging to three main types of farms: breeding, fattening and farrow-to-finish. HEV-RNA was analyzed by broad range Real-time RT-PCR on 20 samples for each farm, obtained by pooling together feces from 10 individuals. Overall, HEV RNA was confirmed on 150 fecal pooled samples out of 1,032 (14.5%). At least one positive pooled sample was detected from 18 farms out of 51 tested (35.3%). By lowering the number of infected pigs at primary production, the risk of HEV-3 entering into the food chain can be reduced. Hence, information on HEV circulation in herds is highly relevant for choosing preventive measures and deserves development of a monitoring program and further investigations.

Two unrelated cases with biallelic CHEK2 variants:a novel condition with constitutional chromosomal instability?

Constitutional heterozygous mutations in CHEK2 gene have been associated with hereditary cancer risk. To date, only a few homozygous CHEK2 mutations have been reported in families with cancer susceptibility. Here, we report two unrelated individuals with a personal and familial cancer history in whom biallelic CHEK2 alterations were identified. The first case resulted homozygous for the CHEK2 c.793-1 G > A (p.Asp265Thrfs*10) variant, and the second one was found to be compound heterozygous for the c.1100delC (p.Thr367Metfs*15) and the c.1312 G > T (p.Asp438Tyr) variants. Multiple cytogenetic anomalies were demonstrated on peripheral lymphocytes of both patients. A literature revision showed that a single other CHEK2 homozygous variant was previously associated to a constitutional randomly occurring multi-translocation karyotype from peripheral blood in humans. We hypothesize that, at least some biallelic CHEK2 mutations might be associated with a novel disorder, further expanding the group of chromosome instability syndromes. Additional studies on larger cohorts are needed to confirm if chromosomal instability could represent a marker for CHEK2 constitutionally mutated recessive genotypes, and to investigate the cancer risk and the occurrence of other anomalies typically observed in chromosome instability syndromes.

What is a biosecurity measure? A definition proposal for animal production and linked processing operations.

While biosecurity, a central component of the One Health concept, is clearly defined, a harmonized definition of the term ´biosecurity measure´ (BSM) is missing. In turn, particularly at the farm and policy level, this leads to misunderstandings, low acceptance, poor implementation, and thus suboptimal biosecurity along the food animal production chain. Moreover, different views on BSMs affects making comparisons both at the policy level as well as in the scientific community. Therefore, as part of the One Health EJP BIOPIGEE project, a work group i) collected and discussed relevant inclusion and exclusion criteria for measures to be considered in the context of biosecurity and ii) conducted a systematic literature review for potentially existing definitions for the term BSM. This exercise confirmed the lack of a definition of BSM, underlining the importance of the topic. In the pool of articles considered relevant to defining the term BSM, specific research themes were identified. Based on these outcomes, we propose a definition of the term BSM: "A biosecurity measure (BSM) - is the implementation of a segregation, hygiene, or management procedure (excluding medically effective feed additives and preventive/curative treatment of animals) that specifically aims at reducing the probability of the introduction, establishment, survival, or spread of any potential pathogen to, within, or from a farm, operation or geographical area." The definition provides a basis for policymakers to identify factual BSMs, highlights the point of implementation and supports to achieve the necessary quality standards of biosecurity in food animal production. It also enables clear, harmonized, cross-sectoral communication of best biosecurity practices to and from relevant stakeholders and thus contribute to improving biosecurity and thereby strengthen the One Health approach.

Bacteriophage Therapy to Reduce Colonization of Campylobacter jejuni in Broiler Chickens before Slaughter.

Campylobacteriosis is the most commonly reported gastrointestinal disease in humans. Campybacter jejuni is the main cause of the infection, and bacterial colonization in broiler chickens is widespread and difficult to prevent, leading to high risk of occurrence in broiler meat. Phage therapy represents an alternative strategy to control Campylobacter in poultry. The aim of this work was to assess the efficacy of two field-isolated bacteriophages against experimental infections with an anti-microbial resistant (AMR) Campylobacter jejuni strain. A two-step phage application was tested according to a specific combination between chickens' rearing time and specific multiplicities of infections (MOIs), in order to reduce the Campylobacter load in the animals at slaughtering and to limit the development of phage-resistant mutants. In particular, 75 broilers were divided into three groups (A, B and C), and phages were administered to animals of groups B and C at day 38 (Φ 16-izsam) and 39 (Φ 7-izsam) at MOI 0.1 (group B) and 1 (group C). All broilers were euthanized at day 40, and Campylobacter jejuni was enumerated in cecal contents. Reductions in Campylobacter counts were statistically significant in both group B (1 log10 colony forming units (cfu)/gram (gr)) and group C (2 log10 cfu/gr), compared to the control group. Our findings provide evidence about the ability of phage therapy to reduce the Campylobacter load in poultry before slaughtering, also associated with anti-microbial resistance pattern.

Characterization and In Vitro Efficacy against Listeria monocytogenes of a Newly Isolated Bacteriophage, ɸIZSAM-1.

Listeria monocytogenes is a bacterial pathogen responsible of listeriosis, a disease that in humans is often related to the contamination of ready-to-eat foods. Phages are candidate biodecontaminants of pathogenic bacteria thanks to their ability to lyse prokaryotes while being safe for eukaryotic cells. In this study, ɸIZSAM-1 was isolated from the drain-waters of an Italian blue cheese plant and showed lytic activity against antimicrobial resistant Listeria monocytogenes strains. This phage was subjected to purification and in vitro efficacy tests. The results showed that at multiplicities of infection (MOIs) ≤ 1, phages were able to keep Listeria monocytogenes at low optical density values up to 8 h, with bacterial counts ranging from 1.02 to 3.96 log10 units lower than the control. Besides, ɸIZSAM-1 was further characterized, showing 25 principal proteins (sodium dodecyl sulfate polyacrylamide gel electrophoresis profile) and a genome of approximately 50 kilo base pairs. Moreover, this study describes a new approach to phage isolation for applications in Listeriamonocytogenes biocontrol in food production. In particular, the authors believe that the selection of phages from the same environments where pathogens live could represent a new approach to successfully integrating the control measures in an innovative, cost effective, safe and environmentally friendly way.

The role of staff and contaminated environmental surfaces in spreading of norovirus infection in a long-term health care facility in Italy.

Some residents and people from the staff of a geriatric health care facility in Teramo province, developed acute gastroenteritis from March 8th to March 21st 2017. A prompt epidemiological investigation was conducted to identify the etiological agent, the trace back the potential ways of transmission and control the infection. Information on the outbreak was collected through an epidemiological questionnaire. Faecal samples from all human cases (n = 50) and swabs from environmental surfaces were collected and analysed by RT-PCR for the presence of Norovirus (NoV). Among faecal samples, 34 out of 50 were positive for NoV with no other pathogen detected. In particular, 2 (2/34) were positive to NoV genogroup I (GI), 31 (31/34) to NoV genogroup II (GII), and one sample (1/34) was positive to both NoV GI and GII. Moreover, faecal samples of people from the canteen (n = 8) were also tested resulting negative to NoV detection. Norovirus was also detected in 28 of the 122 swabs from environmental surfaces collected. Among the positive samples, 12 NoV strains were subtyped as NoV GII.4 Sydney_2012 variant. Person-to-person close contact and contaminated environmental surfaces were the probable transmission route among the people of the health care facility. The members of the staff were considered to play an important role in transmission of NoV. A proper disinfection procedure applied during the outbreak could have been critically important to limit the dissemination of the viral infection.

A Single Center Retrospective Review of Patients from Central Italy Tested for Melanoma Predisposition Genes.

BACKGROUND: Cutaneous malignant melanoma (CMM) is one of the most common skin cancers worldwide. CMM pathogenesis involves genetic and environmental factors. Recent studies have led to the identification of new genes involved in CMM susceptibility: beyond CDKN2A and CDK4, BAP1, POT1, and MITF were recently identified as potential high-risk melanoma susceptibility genes. OBJECTIVE: This study is aimed to evaluate the genetic predisposition to CMM in patients from central Italy. METHODS: From 1998 to 2017, genetic testing was performed in 888 cases with multiple primary melanoma and/or familial melanoma. Genetic analyses included the sequencing CDKN2A, CDK4, BAP1, POT1, and MITF in 202 cases, and of only CDKN2A and CDK4 codon 24 in 686 patients. By the evaluation of the personal and familial history, patients were divided in two clinical categories: "low significance" and "high significance" cases. RESULTS: 128 patients (72% belonging to the "high significance" category, 28% belonging to the "low significance" category) were found to carry a DNA change defined as pathogenic, likely pathogenic, variant of unknown significance (VUS)-favoring pathogenic or VUS. CONCLUSIONS: It is important to verify the genetic predisposition in CMM patients for an early diagnosis of further melanomas and/or other tumors associated with the characterized genotype.

Whole Genome Sequencing Characterization of HEV3-e and HEV3-f Subtypes among the Wild Boar Population in the Abruzzo Region, Italy: First Report.

Hepatitis E virus (HEV) is an emergent zoonotic pathogen, causing worldwide acute and chronic hepatitis in humans. HEV comprises eight genotypes and several subtypes. HEV genotypes 3 and 4 (HEV3 and HEV4) are zoonotic. In Italy, the most part of HEV infections (80%) is due to autochthonous HEV3 circulation of the virus, and the key role played by wild animals is generally accepted. Abruzzo is an Italian region officially considered an HEV "hot spot", with subtype HEV3-c being up to now the only one reported among wild boars. During the year 2018-2019, a group of wild boars in Abruzzo were screened for HEV; positive RNA liver samples were subjected to HEV characterization by using the whole genome sequencing (WGS) approach methodology. This represents the first report about the detection of HEV-3 subtypes e and f in the wild boar population in this area. Since in Italy human infections from HEV 3-e and f have been associated with pork meat consumption, our findings deserve more in-depth analysis with the aim of evaluating any potential correlation between wild animals, the pork chain production and HEV human infections.

Campylobacter bacteriophage DA10: an excised temperate bacteriophage targeted by CRISPR-cas.

BACKGROUND: Lytic bacteriophages that infect Campylobacter spp. have been utilized to develop therapeutic/decontamination techniques. However, the association of Campylobacter spp. and bacteriophages has been the focus of several strands of research aimed at understanding the complex relationships that have developed between predators and prey over evolutionary time. The activities of endogenous temperate bacteriophages have been used to evaluate genomic rearrangements and differential protein expression in host cells, and mechanisms of resistance to bacteriophage infection in campylobacters such as phase variation and CRISPR-mediated immunity. RESULTS: Temperate bacteriophage DA10 represents a novel excised and infective virus capable of replication in a restricted set of C. jejuni and C. coli hosts. Whole genome sequencing reveals that DA10 (35,379 bp) forms part of a novel group of temperate bacteriophages that have limited distribution among database host genome sequences. Analysis of potential host genomes reveals a robust response against DA10 and DA10-like bacteriophages is driven by CRISPR-mediated immunity with 75% of DA10 ORFs represented as ~ 30 bp spacer sequences in numerous Campylobacter Type II-C CRISPR arrays. Several DA10-like homologues have been identified in a small sub-set of C. jejuni and C. coli genome sequences (ranging from near complete integrated prophage sequences to fragments recognisable in the sequence read archive). CONCLUSIONS: A complete intact DA10-like prophage in C. jejuni CJ677CC520 provides evidence that the associations between host and DA10-like bacteriophages are long-standing in evolutionary timescales. Extensive nucleotide substitution and loss can be observed in the integrated DA10-like prophage of CJ677CC520 compared to other relatives as observed through pairwise genome comparisons. Examining factors that have limited the population expansion of the prophage, while others appear to have thrived and prospered (Mu-like, CJIE-like, and lytic Campylobacter bacteriophages) will assist in identifying the underlying evolutionary processes in the natural environment.

A novel LAMP2 mutation associated with severe cardiac hypertrophy and microvascular remodeling in a female with Danon disease: a case report and literature review.

BACKGROUND: Danon disease (DD) is a rare disorder characterized by cardiomyopathy, intellectual disability, and proximal myopathy. It is caused by mutations in the LAMP2 gene on X chromosome. Female patients most often present with late-onset cardiomyopathy and slow disease progression, but early-onset cases with unfavorable prognosis have been reported. CASE REPORT: We describe the clinical, pathological, and molecular features of a novel LAMP2 c.453delT mutation in a female patient with severe hypertrophic cardiomyopathy, Wolff Parkinson White (WPW) syndrome and rapid progression to heart failure, requiring heart transplant. Immunohistochemical analysis of LAMP2 in the explanted heart revealed a mosaic pattern of distribution, with discrete clusters of either stained or unstained cardiac myocytes, the latter being more frequent in the septum. These findings paralleled X chromosome inactivation within the myocardium. Interestingly, multiple foci of microscarring were found on histology in the Left Ventricle (LV) free wall and septum, in a close spatial relationship with remodeling and severe stenosis of intramural coronary arterioles. CONCLUSIONS: Our findings suggest that several features may contribute to the early and severe cardiac phenotype in female DD patients. The type of mutation may account for the early disease onset, while both the inhomogeneous distribution of LAMP2 loss and the presence of microvascular remodeling may be determinant in the rapid progression to heart failure.

Prediction and visualization data for the interpretation of sarcomeric and non-sarcomeric DNA variants found in patients with hypertrophic cardiomyopathy.

Genomic technologies are redefining the understanding of genotype-phenotype relationships and over the past decade, many bioinformatics algorithms have been developed to predict functional consequences of single nucleotide variants. This article presents the data from a comprehensive computational workflow adopted to assess the biomedical impact of the DNA variants resulting from the experimental study "Molecular analysis of sarcomeric and non-sarcomeric genes in patients with hypertrophic cardiomyopathy" (Bottillo et al., 2016) [1]. Several different independently methods were employed to predict the functional consequences of alleles that result in amino acid substitutions, to study the effect of some DNA variants over the splicing process and to investigate the impact of a sequence variant with respect to the evolutionary conservation.

Molecular analysis of sarcomeric and non-sarcomeric genes in patients with hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a common genetic heart disorder characterized by unexplained left ventricle hypertrophy associated with non-dilated ventricular chambers. Several genes encoding heart sarcomeric proteins have been associated to HCM, but a small proportion of HCM patients harbor alterations in other non-sarcomeric loci. The variable expression of HCM seems influenced by genetic modifier factors and new sequencing technologies are redefining the understanding of genotype-phenotype relationships, even if the interpretations of the numerous identified variants pose several challenges. METHODS AND RESULTS: We investigated 62 sarcomeric and non-sarcomeric genes in 41 HCM cases and in 3 HCM-related disorders patients. We employed an integrated approach that combines multiple tools for the prediction, annotation and visualization of functional variants. Genotype-phenotype correlations were carried out for inspecting the involvement of each gene in age onset and clinical variability of HCM. The 80% of the non-syndromic patients showed at least one rare non-synonymous variant (nsSNV) and among them, 58% carried alterations in sarcomeric loci, 14% in desmosomal and 7% in other non-sarcomeric ones without any sarcomere change. Statistical analyses revealed an inverse correlation between the number of nsSNVs and age at onset, and a relationship between the clinical variability and number and type of variants. CONCLUSIONS: Our results extend the mutational spectrum of HCM and contribute in defining the molecular pathogenesis and inheritance pattern(s) of this condition. Besides, we delineate a specific procedure for the identification of the most likely pathogenetic variants for a next generation sequencing approach embodied in a clinical context.

A 22-Week-Old Fetus with Nager Syndrome and Congenital Diaphragmatic Hernia due to a Novel SF3B4 Mutation.

Nager syndrome, or acrofacial dysostosis type 1 (AFD1), is a rare multiple malformation syndrome characterized by hypoplasia of first and second branchial arches derivatives and appendicular anomalies with variable involvement of the radial/axial ray. In 2012, AFD1 has been associated with dominant mutations in SF3B4. We report a 22-week-old fetus with AFD1 associated with diaphragmatic hernia due to a previously unreported SF3B4 mutation (c.35-2A>G). Defective diaphragmatic development is a rare manifestation in AFD1 as it is described in only 2 previous cases, with molecular confirmation in 1 of them. Our molecular finding adds a novel pathogenic splicing variant to the SF3B4 mutational spectrum and contributes to defining its prenatal/fetal phenotype.