Identification of IncA Plasmid, Harboring blaVIM-1 Gene, in S. enterica Goldcoast ST358 and C. freundii ST62 Isolated in a Hospitalized Patient.

In the present study, we analyzed the genome of two S. enterica strains TS1 and TS2 from stool and blood cultures, respectively, and one strain of C. freundii TS3, isolated from a single hospitalized patient with acute myeloid leukemia. The S. enterica Goldcoast ST358 (O:8 (C2-C3) serogroup), sequenced by the MiSeq Illumina system, showed the presence of ß-lactamase genes (blaVIM-1, blaSHV-12 and blaOXA-10), aadA1, ant(2″)-Ia, aac(6')-Iaa, aac(6')-Ib3, aac(6')-Ib-cr, qnrVC6, parC(T57S), and several incompatibility plasmids. A wide variety of insertion sequences (ISs) and transposon elements were identified. In C. freundii TS3, these were the blaVIM-1, blaCMY-150, and blaSHV-12, aadA1, aac(6')-Ib3, aac(6')-Ib-cr, mph(A), sul1, dfrA14, ARR-2, qnrVC6, and qnrB38. IncA plasmid isolated from E.coli/K12 transconjugant and C. freundii exhibited a sequence identity >99.9%. The transfer of IncA plasmid was evaluated by conjugation experiments.

Evaluation of next generation sequencing approaches for SARS-CoV-2.

Within public health control strategies for SARS-CoV-2, whole genome sequencing (WGS) is essential for tracking viral spread and monitoring the emergence of variants which may impair the effectiveness of vaccines, diagnostic methods, and therapeutics. In this manuscript different strategies for SARS-CoV-2 WGS including metagenomic shotgun (SG), library enrichment by myBaits® Expert Virus-SARS-CoV-2 (Arbor Biosciences), nCoV-2019 sequencing protocol, ampliseq approach by Swift Amplicon® SARS-CoV-2 Panel kit (Swift Biosciences), and Illumina COVIDSeq Test (Illumina Inc.), were evaluated in order to identify the best approach in terms of results, labour, and costs. The analysis revealed that Illumina COVIDSeq Test (Illumina Inc.) is the best choice for a cost-effective, time-consuming production of consensus sequences.

Spatial and temporal dynamics of West Nile virus between Africa and Europe.

It is unclear whether West Nile virus (WNV) circulates between Africa and Europe, despite numerous studies supporting an African origin and high transmission in Europe. We integrated genomic data with geographic observations and phylogenetic and phylogeographic inferences to uncover the spatial and temporal viral dynamics of WNV between these two continents. We focused our analysis towards WNV lineages 1 (L1) and 2 (L2), the most spatially widespread and pathogenic WNV lineages. Our study shows a Northern-Western African origin of L1, with back-and-forth exchanges between West Africa and Southern-Western Europe; and a Southern African origin of L2, with one main introduction from South Africa to Europe, and no back introductions observed. We also noticed a potential overlap between L1 and L2 Eastern and Western phylogeography and two Afro-Palearctic bird migratory flyways. Future studies linking avian and mosquito species susceptibility, migratory connectivity patterns, and phylogeographic inference are suggested to elucidate the dynamics of emerging viruses.

Development and validation of an RT-qPCR for detection and quantitation of emerging epizootic hemorrhagic disease virus serotype 8 RNA from field samples.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted virus circulating in multiple serotypes. It has become a concern in the European Union as a novel strain of the serotype 8 (EHDV-8) of clear Northern African origin, has been recently discovered in symptomatic cattle in Italy (islands of Sardinia and Sicily), Spain, and Portugal. Current molecular typing methods targeting the S2 nucleotide sequences -coding for the outermost protein of the virion VP2- are not able to detect the novel emerging EHDV-8 strain as they enrolled the S2 sequence of the unique EHDV-8 reference strain isolated in Australia in 1982. Thus, in this study, we developed and validated a novel typing assay for the detection and quantitation of the novel EHDV-8 RNA from field samples, including blood of ruminants and insects. This molecular tool will certainly support EHDV-8 surveillance and control.

Tick Species Diversity and Molecular Identification of Spotted Fever Group Rickettsiae Collected from Migratory Birds Arriving from Africa.

The role of migratory birds in the spread of ticks and tick-borne pathogens along their routes from Africa to Europe is increasingly emerging. Wild birds can host several tick species, often infected by bacteria responsible for zoonoses. The aim of the study is to assess the possible introduction of exotic ticks carried by migratory birds into Italy from Africa and to detect the presence of Rickettsia species and Coxiella burnetii they may harbor. During a two-year survey, we collected ticks from migratory birds captured during their short stop-over on Ventotene Island. Specimens were first identified by morphology or sequencing molecular targets when needed, and then tested by real-time PCR for the presence of selected pathogens. A total of 91% of the collection consisted of sub-Saharan ticks, more than 50% of which were infected by Rickettsia species belonging to the spotted fever group, mainly represented by R. aeschlimannii. In contrast, the suspected C. burnetii detected in two soft ticks were confirmed as Coxiella-like endosymbionts and not the pathogen. Although there are still gaps in the knowledge of this dispersal process, our findings confirm the role of migratory birds in the spread of ticks and tick-borne pathogens, suggesting the need for a continuous surveillance to monitor the potential emergence of new diseases in Europe.

Genomic and Antimicrobial Surveillance of Campylobacter Population in Italian Poultry.

Campylobacter is one of the most common foodborne diseases worldwide with increasing rates of antibiotic resistance. Most cases of campylobacteriosis can be traced back to the consumption of poultry meat. Despite many efforts to reduce contamination in farms and in slaughterhouses, the persistence of this pathogen in poultry products remains a problem. This study aimed to evaluate the genetic diversity and antibiotic resistance of 542 C. jejuni and C. coli in Italian poultry, in the framework of two National Monitoring Programs. Genomes were screened for antibiotic resistance, virulence determinants and contextualized within a global collection of C. jejuni. ST2116, ST2863 and ST 832 were the most prevalent and significantly associated with Italian poultry. A worrying increase in resistance to quinolones, fluoroquinolones and tetracycline was observed in C. jejuni, while an increased occurrence of multidrug resistant (MDR) strains and strains resistant to macrolides was detected in C. coli. Low resistance rates were found for aminoglycosides. Molecular resistance determinants were consistent with the phenotypic resistance for tetracycline and quinolones. In silico analysis revealed 119 genes associated with virulence factors, with a notably higher prevalence of some genes in ST2863 genomes. This study highlights the increased resistance to macrolides and the emergence of MDR strains for C. coli, the genetic basis of AMR and the predominance of two genotypes among Campylobacter strains isolated from the Italian poultry farms.

Novel Amplicon-Based Sequencing Approach to West Nile Virus.

West Nile virus is a re-emerging arbovirus whose impact on public health is increasingly important as more and more epidemics and epizootics occur, particularly in America and Europe, with evidence of active circulation in Africa. Because birds constitute the main reservoirs, migratory movements allow the diffusion of various lineages in the world. It is therefore crucial to properly control the dispersion of these lineages, especially because some have a greater health impact on public health than others. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to West Nile virus. This study was carried out on different strains from lineage 1 and 2 from Senegal and Italy. The presented protocol/approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for West Nile genomic surveillance.

Severe Streptococcus equi Subspecies zooepidemicus Outbreak from Unpasteurized Dairy Product Consumption, Italy.

During November 2021-May 2022, we identified 37 clinical cases of Streptococcus equi subspecies zooepidemicus infections in central Italy. Epidemiologic investigations and whole-genome sequencing showed unpasteurized fresh dairy products were the outbreak source. Early diagnosis by using sequencing technology prevented the spread of life-threatening S. equi subsp. zooepidemicus infections.

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.

Metagenomic Characterization and Volatile Compounds Determination in Rumen from Saanen Goat Kids Fed Olive Leaves.

The accumulation and disposal of by-products deriving from the agro-food industry represents a problem both from an economic and environmental point of view. The use of these matrices in zootechnical nutrition could represent a feasible solution. The aim of the study was to examine the effect of a diet containing olive leaves (OL), a by-product of the olive industry, on the ruminal microbial community of Saanen goat kids and on volatile organic compounds (VOCs) produced during the digestion. Twenty goat kids were randomly divided into two groups of ten goat kids each. The control group (CTR) was fed with a standard diet, while the experimental group (OL+) received a custom-formulated diet containing 10 % OL on a dry matter (DM) basis. After 30 days of trial, genomic DNA was extracted from the rumen liquor and prepared for 16S rRNA-gene sequencing to characterize the rumen microbiota; furthermore, rumen VOCs were also characterized by solid-phase microextraction coupled with gas chromatography-mass spectrometry. The Shannon's alpha index was not significantly different between the two groups, on the contrary, Bray-Curtis (p < 0.01) and Jaccard (p < 0.01) distances evidenced that feed affected microbial community. Eleven genera were influenced by OL supplementation, with a significant increase (p < 0.05) in Paludibacter, Fibrobacter, Sphaerochaeta Christensenella, Rikenella, Oligosphaera, Candidatus Endomicrobium, Anaerovorax, and Atopobium was observed, while the percentages of Bacteroides and Selenomonas were reduced (p < 0.05). Differences were also observed between the two groups at the family level (p < 0.004). Fibrobacteriaceae, Christensenellaceae, Coriobacteriaceae, Oligosphaeraceae, Candidatus Endomicrobium, and Planctomycetaceae were significantly higher (p < 0.05) in goat kids fed OL diet compared to CTR, while the levels of other identified families, Succinivibrionaceae and Bifidobacteriaceae, were opposite (p < 0.05). Finally, results showed that the main phyla in both groups were Bacteroidetes and Firmicutes; however, no significant differences in the relative abundance of any phyla were observed between the two groups. In addition to what has been reported, the analysis of VOCs at the rumen level showed the ability of the OL integration to induce an increase in hexanoic acid and a parallel decrease in decanal. Furthermore, only in OL+ samples there was the accumulation of α-terpineol to which a wide range of interesting biological properties is attributed. The presence of VOCs associated with health status suggests a favorable role of OL in preserving and improving animal welfare.

Validation of AmpliSeq NGS Panel for BRCA1 and BRCA2 Variant Detection in Canine Formalin-Fixed Paraffin-Embedded Mammary Tumors.

Mammary carcinomas are the most common neoplasms observed in women and in female dogs. Canine mammary tumors show epidemiological, clinical, genetic, and prognostic characteristics comparable to human breast cancers. The recent introduction of next generation sequencing (NGS) technologies has greatly improved research and diagnostics for humans, while these new tools still need to be implemented in animal models. In this study we developed and validated an AmpliSeq Panel assay for the identification of BRCA variants in twenty-two different dogs. The amplicon mean coverage was 5499× and uniformity was higher than 98% in all samples. The results of germline single nucleotide variants (SNVs) and insertions/deletions (INDELs) were fully concordant regardless of the types of samples considered (blood, fresh and FFPE tissues). Moreover, despite the high DNA degradation observed in older FFPE blocks (>5 years), the assay allowed full coverage of all amplicons for downstream analyses. We consider the NGS panel developed in this study as a useful tool for expanding information on BRCA genes in the veterinary field and for human health from a comparative oncology perspective.

Impact of different shades of light-emitting diode on fecal microbiota and gut health in broiler chickens.

OBJECTIVE: The aim of this study was to characterize the fecal microbiota of broiler chickens reared in the presence of different shades of light-emitting diode (LED) lights, correlating this information with biochemical and molecular evidence that allowed drawing conclusions on the state of health of the animals. METHODS: Overall, the metagenomic approach on fecal samples was associated with evaluations on enzymes involved in the cellular response to oxidative stress: glutathione peroxidase (GPX), superoxide dismutase and catalase; while the inflammatory aspect was studied through the dosage of a proinflammatory cytokine, the interleukin 6 (IL-6), and the evaluation of the matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9). Specifically, analysis was performed on distinct groups of chickens respectively raised in the presence of neutral (K = 3,300 to 3,700), cool (K = 5,500 to 6,000), and warm (K = 3,000 to 2,500) LED lightings, and a direct comparison was performed with animals reared with traditional neon lights. RESULTS: The metagenomic analysis highlighted the presence of two most abundant bacterial phyla, the Firmicutes and the Bacteroidetes, with the latter characterized by a greater relative abundance (p<0.05) in the group of animals reared with Neutral LED light. The analysis on the enzymes involved in the antioxidant response showed an effect of the LED light, regardless of the applied shade, of reducing the expression of GPX (p<0.01), although this parameter is not correlated to an effective reduction in the tissue amount of the enzyme. Regarding the inflammatory state, no differences associated with IL-6 and MMP-9 were found; however, is noteworthy the significant reduction of MMP-2 activity in tissue samples obtained from animals subjected to illumination with neutral LED light. CONCLUSION: This evidence, combined with the metagenomic findings, supports a potential positive effect of neutral LED lighting on animal welfare, although these considerations must be reflected in more targeted biochemical evaluations.

Serological, Molecular Prevalence and Genotyping of Coxiella burnetii in Dairy Cattle Herds in Northeastern Algeria.

In Algeria, data on the epidemiology of coxiellosis in cattle are still lacking. In this study, bulk tank milk (BTM) samples from 200 randomly selected dairy cattle herds from Setif province of Algeria were analyzed by an indirect enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Results highlighted that 37% (95% CI: 30.31-43.69%) and 9% (95% CI: 5.03-12.96%) of BTM samples contained Coxiella burnetii antibodies and DNA, respectively. Based on Cohen's kappa coefficient, a very low agreement between the ELISA and PCR results was found (k = 0.0849) (95% CI: 0.00-0.189). For a second experiment, 186 whole blood samples of cows from farms with reproduction disorders were analyzed by molecular tools to detect C. burnetii. This study revealed an overall prevalence of 6.98% (95% CI: 3.32-10.65%). All positive samples determined by conventional PCR were analyzed by real-time quantitative PCR (qPCR). Eleven samples with cycle threshold (Ct) values lower than 35 were selected for genotyping by the multispacer sequence typing (MST) method. The MST12 genotype in BTM samples, the MST32 genotype and a new MST genotype (partial profile) in whole blood samples were identified. Obtained results have allowed us to better understand the epidemiology of bovine coxiellosis in the region of Setif.

Phylogenetic Analysis and Genome-Wide Association Study Applied to an Italian Listeria monocytogenes Outbreak.

From May 2015 to March 2016, a severe outbreak due to Listeria monocytogenes ST7 strain occurred in Central Italy and caused 24 confirmed clinical cases. The epidemic strain was deeply investigated using whole-genome sequencing (WGS) analysis. In the interested area, the foodborne outbreak investigation identified a meat food-producing plant contaminated by the outbreak strain, carried by pork-ready-to-eat products. In the same region, in March 2018, the epidemic strain reemerged causing one listeriosis case in a 10-month-old child. The aim of this study was to investigate the phylogeny of the epidemic and reemergent strains over time and to compare them with a closer ST7 clone, detected during the outbreak and with different pulsed-field gel electrophoresis (PFGE) profiles, in order to identify genomic features linked to the persistence and the reemergence of the outbreak. An approach combining phylogenetic analysis and genome-wide association study (GWAS) revealed that the epidemic and reemergent clones were genetically closer to the ST7 clone with different PFGE profiles and strictly associated with the pork production chain. The repeated detection of both clones was probably correlated with (i) the presence of truly persistent clones and the repeated introduction of new ones and (ii) the contribution of prophage genes in promoting the persistence of the epidemic clones. Despite that no significant genomic differences were detected between the outbreak and the reemergent strain, the two related clones detected during the outbreak can be differentiated by transcriptional factor and phage genes associated with the phage LP-114.

Congenital Oral Squamous Cell Carcinoma in a Suckling Piglet.

A 3-week-old suckling piglet spontaneously died after septicemic colibacillosis. At postmortem examination, bulging and ulcerated lesions were seen, affecting the oral mucosa on the inner surface of the lower lip. After histopathological investigation, the diagnosis of congenital oral squamous cell carcinoma was made. To the best of our knowledge, this is the first case of congenital oral squamous cell carcinoma ever described. A relationship has been shown or suggested between papillomavirus infection and oral squamous cell carcinoma in humans and animals. However, next-generation sequencing study did not demonstrate any papillomavirus sequences in the case reported herein.

Whole-Genome Sequences of SARS-CoV-2 Lineage B.1.525 Strains (Variant η) Detected from Patients in the Abruzzo Region (Central Italy) during Spring 2021.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are emerging worldwide. Here, we report the complete genome sequences of 13 severe acute SARS-CoV-2 strains belonging to lineage B.1.525 (variant η).

Intensive Environmental Surveillance Plan for Listeria monocytogenes in Food Producing Plants and Retail Stores of Central Italy: Prevalence and Genetic Diversity.

Listeria monocytogenes (Lm) can persist in food processing environments (FPEs), surviving environmental stresses and disinfectants. We described an intensive environmental monitoring plan performed in Central Italy and involving food producing plants (FPPs) and retail grocery stores (RSs). The aim of the study was to provide a snapshot of the Lm circulation in different FPEs during a severe listeriosis outbreak, using whole genome sequencing (WGS) to investigate the genetic diversity of the Lm isolated, evaluating their virulence and stress resistance profiles. A total of 1217 samples were collected in 86 FPEs with 12.0% of positive surfaces at FPPs level and 7.5% at RSs level; 133 Lm isolates were typed by multilocus sequencing typing (MLST) and core genome MLST (cgMLST). Clonal complex (CC) 121 (25.6%), CC9 (22.6%), CC1 (11.3%), CC3 (10.5%), CC191 (4.5%), CC7 (4.5%) and CC31 (3.8%) were the most frequent MLST clones. Among the 26 cgMLST clusters obtained, 5 of them persisted after sanitization and were re-isolated during the follow-up sampling. All the CC121 harboured the Tn6188_qac gene for tolerance to benzalkonium chloride and the stress survival islet SSI-2. The CC3, CC7, CC9, CC31 and CC191 carried the SSI-1. All the CC9 and CC121 strains presented a premature stop codon in the inlA gene. In addition to the Lm Pathogenicity Island 1 (LIPI-1), CC1, CC3 and CC191 harboured the LIPI-3. The application of intensive environmental sampling plans for the detection and WGS analysis of Lm isolates could improve surveillance and early detection of outbreaks.

Draft Genome Sequence of Serratia rubidaea, a Potential Opportunistic Pathogen Isolated from Food in Italy.

Serratia rubidaea has emerged in recent years as an opportunistic nosocomial pathogen. Here, we present the draft genome sequence of an isolate derived from an industrial meat food product purchased in a large-scale retail store that revealed fluoroquinolone, ß-lactam, and aminoglycoside resistance genes and two different host-unspecific prophages.

Infection sustained by lineage B.1.1.7 of SARS-CoV-2 is characterised by longer persistence and higher viral RNA loads in nasopharyngeal swabs.

Following the announcement on December 2020 about the emergence of a new variant (VOC 202012/ 01, B.1.1.7 lineage) in the United Kingdom, a targeted surveillance was put in place in the Abruzzo region (Italy), which allowed detection of 313 persons affected by lineage B.1.1.7, up to the 20th of February 2021. We investigated the results of RT-PCR on nasopharyngeal swabs tested from December 2020 to February 2021 to verify any difference on the viral load and persistence between people infected by lineage B.1.1.7 and others. Statistically significant lower values of CT associated with the detection of the N protein encoding gene (CT N) were observed in persons with lineage B.1.1.7 infection (median CT N = 15.8)in comparison to those infected by other lineages (median CT N = 16.9). A significantly longer duration of the persistence of SARS-CoV-2 RNA in nasopharyngeal swabs was observed in persons with lineage B.1.1.7 infection (16 days) in comparison to those infected by other lineages (14 days).

A large food-borne outbreak of campylobacteriosis in kindergartens and primary schools in Pescara, Italy, May-June 2018.

Introduction. In May-June 2018, an outbreak of campylobacteriosis involved students and school staff from kindergartens and primary schools in Pescara, southern Italy.Aim. We present details of the epidemiological and microbiological investigation, and the findings of the analytical study, as well as the implemented control measures.Methodology. To identify possible risk factors associated with the observed outbreak, a case control study was conducted using a questionnaire to collect information on the date of symptoms onset, type and duration of symptoms, type of healthcare contact, school attendance, and food items consumed at school lunches during the presumed days of exposure. Attack rates were calculated for each date and school. Logistic regression models were used to estimate the odds ratios of being a case and the odds of illness by food items consumed, respectively. Moreover, we carried out a comparative genomic analysis using whole genome multilocus sequence typing (wgMLST) of Campylobacter jejuni strains isolated during the outbreak investigation to identify the source of the outbreak.Results. Overall, 222 probable cases from 21 schools were identified, and C. jejuni was successfully isolated from 60 patients. The meals in the schools involved were provided by two cooking centres managed by a joint venture between two food companies. Environmental and food sampling, epidemiological and microbiological analyses, as well as a case control study with 176 cases and 62 controls from the same schools were performed to identify the source of the outbreak. The highest attack rate was recorded among those having lunch at school on 29 May (7.8 %), and the most likely exposure was 'caciotta' cheese (odds ratio 2.40, 95 % confidence interval 1.10-5.26, P=0.028). C. jejuni was isolated from the cheese, and wgMLST showed that the human and cheese isolates belonged to the same genomic cluster, confirming that the cheese was the vehicle of the infection.Conclusion. It is plausible that a failure of the pasteurization process contributed to the contamination of the cheese batches. Timely suspension of the catering service and summer closure of the schools prevented further spread.