Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise G. Caporale

Two new quantitative methods for the determination of four stress-related hormones (cortisol, cortisone, dehydroepiandrosterone and dehydroepiandrosterone sulfate) in horse hair and sheep wool by liquid chromatography coupled with hybrid high resolution mass spectrometry were developed and validated. Hormones were extracted overnight from ground samples with methanol after a washing step with the same solvent to remove external contamination. The extract was evaporated and dry residue was solubilized in an acid mixture of methanol and water. The methods were validated according to ICH guidelines for bioanalytical method validation within 5–100 pg/mg with LC-Q Exactive platform (except for DHEA: 100–2000 pg/mg for both matrices and 1–100 pg/mg for DHEAS in mane hair). The validated concentration range was 5–100 pg/mg for wool and 1–100 pg/mg (except for DHEA: 5–100 pg/mg) for mane hair samples with LC-Exploris 120 system. Satisfactory quantitative performances were obtained using isotopic dilution and surrogate analyte approach. After successful validation, the applicability of the methods were tested. The detected concentrations of investigated analytes in real samples collected from animals living in marginal areas were encompassing the interval 1.1–13 and 5.0–30.5 pg/mg in mane hair and wool samples, respectively.

Different laboratories employ different Whole-Genome Sequencing (WGS) pipelines for Food and Waterborne disease (FWD) surveillance, casting doubt on the comparability of their results and hindering optimal communication at intersectoral and international levels. Through a collaborative effort involving eleven European institutes spanning the food, animal, and human health sectors, we aimed to assess the inter-pipeline clustering congruence across all resolution levels and perform an in-depth comparative analysis of cluster composition at outbreak level for four important foodborne pathogens: Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Campylobacter jejuni. We found a general concordance between allele-based pipelines for all species, except for C. jejuni, where the different resolution power of allele-based schemas led to marked discrepancies. Still, we identified non-negligible differences in outbreak detection and demonstrated how a threshold flexibilization favors the detection of similar outbreak signals by different laboratories. These results, together with the observation that different traditional typing groups (e.g., serotypes) exhibit a remarkably different genetic diversity, represent valuable information for future outbreak case-definitions and WGS-based nomenclature design. This study reinforces the need, while demonstrating the feasibility, of conducting continuous pipeline comparability assessments, and opens good perspectives for a smoother international and intersectoral cooperation towards an efficient One Health FWD surveillance.

In this study, two cases of sudden death following infection-like symptoms in dairy water buffalo calves aged 5 - 12 days were investigated by anatomopathological examinations and laboratory tests. Four bacterial infectious agents were isolated from the brain, which presented meningitis-like lesions, and liver, which appeared hyperaemic and with fibrin formations. The four isolates were phenotypically identified as Streptococcus suis and found to be genetically identical by whole genome sequencing (WGS). One of the isolates was further characterized by hybrid short and long reads genome sequencing and found to represent a novel sequence type (ST) of S. suis serotype 2 . Further investigations are needed to better understand its pathogenic potential, host specificity and environmental sources of infection.

Listeriosis, caused by Listeria monocytogenes , is a severe foodborne infection, particularly hazardous for pregnant women and immunocompromised individuals. In Europe, L. monocytogenes was the fifth most reported zoonotic agent in 2022, with outbreaks frequently linked to ready‐to‐eat (RTE) foods. Addressing food contamination with innovative antimicrobial strategies is critical to enhancing food safety and reducing public health risks. This study evaluated the efficacy of two bacteriophage‐derived endolysins, LP101_021 and PlyP100, in reducing L. monocytogenes contamination in Squacquerone, a traditional Italian soft cheese made from whole cow's milk. Squacquerone samples were experimentally inoculated with approximately 6 log₁₀ cfu/g of L. monocytogenes strain Lm‐ID11. The samples were then treated with either LP101_021 or PlyP100, and viable bacterial counts were monitored over time. Reductions in bacterial load were measured at 1 and 24 h post treatment to assess the effectiveness of each endolysin. Endolysin LP101_021 demonstrated a significant reduction in L. monocytogenes counts, decreasing bacterial loads by more than 3 log₁₀ cfu/g within 1 h, with sustained effects over 24 h. PlyP100 exhibited a more moderate impact, reducing bacterial counts by approximately 0.5 log₁₀ cfu/g after 1 h and 1.38 log₁₀ cfu/g after 24 h. By comparing the effects of those two endolysins, these findings highlight the rapid and effective antimicrobial action in particular of LP101_021 in cheese matrices. The study underscores the potential of bacteriophage endolysins as targeted antimicrobial agents for food safety applications. Their specificity allows the biocontrol of harmful pathogens without disrupting beneficial microbial communities, making them a promising alternative to conventional antimicrobial strategies. Furthermore, the effectiveness in reducing L. monocytogenes contamination in dairy products supports their potential integration in food processing and storage protocols, addressing the global challenge of antibiotic‐resistant bacteria.

Listeria monocytogenes is an important foodborne pathogen, markedly persistent even in harsh environments and responsible for high hospitalization and mortality rates. The aim of the present study was to detect the strains circulating in Sicily over a five-year period and characterize their antimicrobial resistance profiles. The key element of this study was the sharing of data among various entities involved in food control and clinical surveillance of listeriosis in order to develop an integrated approach for this pathogen. A total of 128 isolates were analyzed, including 87 food-source strains and 41 clinical specimens. Whole-genome sequencing (WGS) was performed for sequence type (ST) and clonal complex (CC) identification through multilocus sequence typing (MLST) analysis. Antimicrobial resistance was assessed using the Kirby–Bauer method. The majority of strains belonged to serotype IVb (34/41 and 53/87 of clinical and food-source isolates, respectively) and were subtyped as CC2-ST2 (28/34 and 41/53 of clinical and food-source isolates respectively). Most of the isolates were susceptible to the main antimicrobials recommended for treatment of listeriosis. Resistance (R) and intermediate resistance (I) percentages worthy of attention were found against oxacillin (R: 85.9%) and clindamycin (I: 34.6%) in the food-source isolates and trimethoprim/sulfamethoxazole (R: 29.23%) in the clinical isolates. Also, 7.7% of the food-source isolates were multidrug resistant. Our results highlight how the punctual comparison between food and clinical strains is an essential tool for effectively tracking and preventing foodborne outbreaks.

Background Bovine coronavirus (BoCV) causes significant economic losses to cattle farming due to mortality in calves, reduced growth performances and milk production in feedlots and dairy cattle. Worldwide distribution of BoCV has been demonstrated, although knowledge of its epidemiology in Africa, especially in the sub-Saharan region, is limited. Results In the present study, a total of 208 swab samples of wild ruminants and 435 bovines from different regions of Namibia were obtained and tested by a BoCV-specific qRT-PCR. Twenty-six bovine samples tested positive [26/435 (5.98%; 95CI: 3.94-8.64%)] while, among the wild ruminants, only Greater Kudu (Tragelaphus strepsiceros) were shown to be positive [13/52 (25.00%; 95CI: 14.03-38.95%)] of which 8 showed clinical signs. Analysis of partial nucleoprotein and spike protein gene sequences and comparison with international reference sequences demonstrated the existence of a unique Namibian clade, resulting from a single introduction event around 2010 followed by local evolution. Although the introduction source remains unknown, contact between bovine and wild animals appears likely. Conclusions The present study represents the first report of BoCV circulation in southern Africa, which showed a relatively high frequency and the ability of persisting and evolving locally in the absence of further foreign introductions. The implications for disease spread among domestic bovines and the potential impact on wildlife should encourage broader investigations on BoCV involving other African countries. Moreover, the Greater Kudu’s susceptibility to BoCV infection was also proven, further highlighting the host plasticity of this virus.

Porcine circovirus 2 (PCV-2) is a major pathogen of swine, causing significant production losses in the pig industry worldwide. Several studies have detected the virus in different species, both in asymptomatic and diseased subjects, highlighting PCV-2 host plasticity. As PCV-2 has been identified in carnivores, the present study was undertaken to investigate the susceptibility of domestic dogs to PCV-2 infection by testing archived blood samples originating from low-income rural areas in Namibia. The population was selected considering the high exposure probability to PCV-2 due to direct contact and/or feeding with raw pig meat or by-products. Thirty-eight of the samples (6.61%) tested positive for PCV-2, and the complete ORF2 of 7 strains was sequenced, revealing the presence of the three major PCV-2 genotypes (i.e. PCV-2a, -2b, and -2d). Convincing epidemiological links with other Namibian and South African strains were established for PCV-2a and PCV-2b strains, while the PCV-2d strains were part of a broader clade that included sequences of viruses collected worldwide, especially in Asia. Although PCV-2 was reported in diseased dogs, no statistically significant or robust causal association between infection and disease manifestation was demonstrated. In conclusion, PCV-2 infection has been identified in Namibian dogs, most likely due to the ingestion of contaminated meat and by-products. However, the epidemiological and clinical consequences are still unclear and further investigations are necessary. Nevertheless, the high proportion of infected dogs showing clinical signs raises concern about the potential of PCV-2’s role as a relevant viral pathogen in Namibia. The use of raw meat for dog nutrition should be discouraged, considering the known risks to animal and human health through disease transmission.

Listeria monocytogenes presents a significant concern for the food industry due to its ability to persist in the food processing environment. One of the factors contributing to its persistence is decreased sensitivity to disinfectants. Our objective was to assess the diversity of L. monocytogenes sensitivity to food industry disinfectants by testing the response of 1,671 L. monocytogenes isolates to quaternary ammonium compounds (QACs) and 414 isolates to peracetic acid (PAA) using broth microdilution and growth curve analysis assays, respectively, and to categorize the isolates into sensitive and tolerant. A high phenotype-genotype concordance (95%) regarding tolerance to QACs was obtained by screening the genomes for the presence of QAC tolerance-associated genes bcrABC, emrE, emrC, and qacH. Based on this high concordance, we assessed the QAC genes’ dissemination among publicly available L. monocytogenes genomes (n = 39,196). Overall, QAC genes were found in 23% and 28% of the L. monocytogenes collection in this study and in the global data set, respectively. bcrABC and qacH were the most prevalent genes, with bcrABC being the most detected QAC gene in the USA, while qacH dominated in Europe. No significant differences (P > 0.05) in the PAA tolerance were detected among isolates belonging to different lineages, serogroups, clonal complexes, or isolation sources, highlighting limited variation in the L. monocytogenes sensitivity to this disinfectant. The present work represents the largest testing of L. monocytogenes sensitivity to important food industry disinfectants at the phenotypic and genomic level, revealing diversity in the tolerance to QACs while all isolates showed similar sensitivity to PAA. IMPORTANCE Contamination of Listeria monocytogenes within food processing environments is of great concern to the food industry due to challenges in eradicating the isolates once they become established and persistent in the environment. Genetic markers associated with increased tolerance to certain disinfectants have been identified, which alongside other biotic and abiotic factors can favor the persistence of L. monocytogenes in the food production environment. By employing a comprehensive large-scale phenotypic testing and genomic analysis, this study significantly enhances the understanding of the L. monocytogenes tolerance to quaternary ammonium compounds (QACs) and the genetic determinants associated with the increased tolerance. We provide a global overview of the QAC genes prevalence among public L. monocytogenes sequences and their distribution among clonal complexes, isolation sources, and geographical locations. Additionally, our comprehensive screening of the peracetic acid (PAA) sensitivity shows that this disinfectant can be used in the food industry as the lack of variation in sensitivity indicates reliable effect and no apparent possibility for the emergence of tolerance.

Plastic marine litter is a critical issue that threatens marine ecosystems. This study investigated microplastics (MPs) contamination in the Italian seas, involving regions significantly affected by pollution from urban, industrial and agricultural sources. The research, conducted in collaborations between 10 different Experimental Zooprophylactic Institutes throughout Italy, analyzed Mytilus galloprovincialis (common mussels) for its filtration capacity and suitability as a bioindicator. Using data from two projects funded by the Italian Ministry of Health, MPs were detected from 7% to 13% of mussel samples, mainly polypropylene and polystyrene fragments and fibers. These findings align with previous studies highlighting the pervasive presence of MPs and their potential risks as mussels are consumed whole, allowing MPs to be ingested. The study underscores the need for standardized detection methods and coordinated policies to mitigate plastic pollution. Public awareness campaigns and improved waste management practices are key to addressing the environmental and health impacts of MPs. Further research on the long-term effects of MPs on marine ecosystems and human health is essential to developing comprehensive mitigation strategies.

The One Health approach unites efforts across human-animal-environment interfaces against shared threats like zoonotic diseases. T-Racing is a Shiny web application, that supports epidemiological investigations and helps contain livestock-related disease spread, aligning with multidisciplinary principles to safeguard public health. The application uses Temporal Network Analysis techniques to address the dynamic nature of animal trade, facilitating backward and forward tracing strategies. T-Racing leverages web services to retrieve data from multiple sources simultaneously and in near real-time through the plumber package and is distributed using Shinyproxy. T-Racing manages and analyze extensive and diverse datasets within the same environment, including animal movement data, disease outbreak data, and genomic data, all obtained from Italian National databases. In this work, we show T-Racing’s capabilities by simulating epidemiological investigations of brucellosis and tuberculosis outbreaks that occurred in non–endemic areas of Italy. To further highlight its capabilities, an interactive demo of T-Racing is available, showcasing its potential and features. This tool supports epidemiological investigations by adopting a data-driven approach, guiding users through the analysis via an iterative process while leveraging their expertise. Therefore, it enables faster data analysis, improves understanding of disease transmission patterns, and facilitates prompt and targeted interventions.

Objectives Human respiratory syncytial virus (hRSV) is the leading cause of severe respiratory infections in children worldwide. Severe cases often require intensive care. Respiratory syncytial virus (RSV) is classified into two main antigenic groups: RSV-A and RSV-B. Recent molecular advancements have significantly enhanced our ability to control and understand RSV infections. This study investigated the epidemiologic and genetic characteristics of the RSV in Tunisia, focusing on its evolutionary dynamics. Methods Between October and December 2021, 92 samples were collected from Tunisian patients hospitalized for mild-to-severe acute respiratory infections. Laboratory analyses, including real-time reverse transcription-polymerase chain reaction and whole genome sequencing (WGS), were performed to identify and characterize the RSV strains. Phylogenetic analyses were performed to compare the Tunisian sequences with the global RSV sequences from 2012 to 2022. Results Of the 92 patients (mean age 1 year and 6 months), 96.9% of the samples tested positive for RSV-A, and 22 samples exhibited co-infections with other respiratory viruses. WGS was successfully performed on the 74 samples. Phylogenetic analysis identified six distinct clades of RSV-A circulating in Tunisia, indicating multiple parallel introductions of the virus into the country. Specific Tunisian clades showed genetic similarities to RSV strains from Argentina, Belgium, and the Philippines. Conclusions This study underscores the genetic diversity of RSV in Tunisia, with multiple introductions after the lifting of COVID-19 restrictions in 2021. WGS revealed significant genetic heterogeneity within RSV-A, which could affect the effectiveness of vaccines and therapeutic strategies. Continued surveillance, particularly, in resource-limited regions, is crucial to inform and guide future interventions.

African swine fever (ASF) is a highly contagious disease affecting wild and domestic pigs, characterised by severe haemorrhagic symptoms and high mortality rates. Originally confined to Sub-Saharan Africa, ASF virus genotype II has spread to Europe since 2014, mainly affecting Eastern Europe, and progressing through wild boar migrations and human action. In January 2022, the first case of ASF, due to genotype II, was reported in North-western Italy, in a wild boar carcass. Thereafter, numerous positive wild boars were identified, indicating an expanding wild epidemic, severely threatening Italian pig farming and trade. This study focused on the mapping of the suitable habitats for wild boars and their potential dispersal corridors in Northern Italy, using species distribution models and landscape connectivity analysis. The resulting maps identified areas with higher likelihood of wild boar presence, highlighting their preferential pathways crossing Northern Italy. The distribution of ASF positive wild boars along the major corridors predicted by the model suggests the obtained maps as valuable support to decision-makers to improve ASF surveillance and carcass early detection, aiming for eradication. The applied framework can be easily replicated in other regions and countries.

MicroRNAs (miRNAs) have acquired an increased recognition to unravel the complex molecular mechanisms underlying Diminished Ovarian Reserve (DOR), one of the main responsible for infertility. To investigate the impact of miRNA profiles in granulosa cells and follicular fluid, crucial players in follicle development, this study employed a computational network theory approach to reconstruct potential pathways regulated by miRNAs in granulosa cells and follicular fluid of women suffering from DOR. Available data from published research were collected to create the FGC_MiRNome_MC, a representation of miRNA target genes and their interactions. 365 hubs were identified within the network, representing potential key regulators, and 210 nodes that act as both hubs and bottlenecks (H&BN nodes), suggesting that they may control the information flow within the network. GO enrichment analysis of the 210 H&BN nodes revealed their involvement in fundamental cellular processes relevant to ovarian function. In particular, the cluster analysis identified several shared pathways between cluster 1 and cluster 2 involved in the RAS/MAPK pathway, which plays a critical role in cell proliferation, differentiation and survival. These findings suggest that miRNAs play a significant role in DOR and highlight the potential of the RAS/MAPK pathway as a target for further investigation. Additionally, the genes identified as both hubs and bottlenecks revealed interesting connections to reproductive health in KO mice models. This in silico approach provides valuable insights into potential biomarkers and therapeutic targets for age-related reproductive disorders.

Members of the RNA virus order Nidovirales infect hosts ranging from marine invertebrates to terrestrial mammals. As such, understanding the determinants of host range in this group of viruses, as well as their patterns of emergence and disease potential, is of clear importance. The Mesoniviridae are a recently documented family within the Nidovirales. To date, mesoniviruses have only been associated with the infection of arthropod species, particularly mosquitoes, and hence are regarded as insect-specific viruses (ISVs). Herein, we report the first detection of a mesonivirus—Alphamesonivirus-1 —in mammals. Specifically, we utilized genomic and histological techniques to identify Alphamesonivirus-1 in lung and lymph node tissues of two horses (a mare and its foal) from Italy that succumbed to an acute respiratory syndrome. The genome sequences of Alphamesonivirus-1 obtained from the two horses were closely related to each other and to those from a local Culex mosquito pool and an Alphamesonivirus-1 previously identified in Italy, indicative of ongoing local transmission. The discovery of Alphamesonivirus-1 in horse tissues prompts further investigation into the host range of mesoniviruses, the possible role of insect-specific viruses in mammalian disease processes, the determinants of and barriers to cross-species virus transmission, and the potential epizootic threats posed by understudied viral families. IMPORTANCE Alphamesoniviruses, members of the family Mesoniviridaeare, are considered insect-specific RNA viruses with no known association with vertebrate hosts. Herein, we report the identification of Alphamesonivirus-1 in mammals. Using detailed molecular and histological analyses, we identified Alphamesonivirus-1 in lung and lymph node tissues of two horses that presented with an acute respiratory syndrome and that was phylogenetically related to virus sequences found in local Culex mosquitoes. Hence, Alphamesoniviruses may possess a broader host range than previously believed, prompting the investigation of their possible role in mammalian disease. This work highlights the need for increased surveillance of atypical viruses in association with unexplained respiratory illness, including those commonly assumed to be insect-specific, and may have implications for epizootic disease emergence.