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This radio-tracking study reports the spring home range, spatial organisation and activity of 11 stoats Mustela erminea in a New Zealand Nothofagus forest, 1.5 yr after significant seedfall when rodent density was low, but stoat density was high. The average home range of 4 male stoats was 223 (SE = 45) ha, significantly larger than the average range area of 94 (SE = 13) ha recorded for 7 female stoats. Stoatswere generally tolerant of sharing space and did not maintain intra-or intersexual territorial spacing systems. There was no evidence oftemporal avoidance with several stoats of the same sex showing slight attraction to one another. However, stoats still may avoid one another when in close proximity. Long-term radio-tracking studies are required to determine the general patterns of spacing behaviour in stoats. Male stoats showed higher levels of activity during daylight than females. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a significant concern in the food industry due to its association with outbreaks of listeriosis, particularly affecting vulnerable populations. High-throughput technologies such as RNA sequencing (RNA-seq) and proteomics offer valuable insights into the molecular responses of L. monocytogenes to stress environments. In this study, a combined transcriptomic and immunoproteomic approach was applied to explore the stress response mechanisms of the L. monocytogenes strain ST7, which was responsible for an outbreak in central Italy. The bacterium was exposed to both optimal conditions and a stress environment representative of pork product matrices (pH 5.5; 7 % NaCl) and thermal abuse prior to consumption (12 °C).Transcriptomic analysis revealed variations in gene expression related to pathogenesis, stress responses, and virulence factors under different environmental conditions. Transcriptomic analysis of Listeria involves studying the complete set of RNA transcripts produced by the bacterium under various conditions or during different stages of its lifecycle. It can provide insights into its pathogenicity and virulence mechanisms. Immunoproteomic analysis identified proteins involved in stress response pathways, including oxidoreductases and DNA repair enzymes, uniquely expressed under stress conditions. Furthermore, the study highlighted proteins linked to antibiotic resistance and cell wall biosynthesis. By delineating specific proteins crucial in the stress response pathways, these findings not only deepen our comprehension of L. monocytogenes biology but also pave the way for designing more targeted mitigation strategies to safeguard food safety and public health effectively., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

Interpreting the phenotypes of bla SHV alleles in Klebsiella pneumoniae genomes is complex. Whilst all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomal bla SHV alleles or additional plasmid-borne bla SHV alleles that have extended-spectrum β-lactamase (ESBL) activity and/or β-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/a changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases [e.g. the National Center for Biotechnology Information (NCBI) Reference Gene Catalog and the β-lactamase database (BLDB)] over the specific functionality of individual sulfhydryl variable (SHV) protein variants. Therefore, the identification of ESBL-producing strains from K. pneumoniae genome data is complicated. Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific aa substitutions. We then systematically assigned SHV alleles to functional classes (WT, ESBL and BLI resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with the current assignments in the NCBI's Reference Gene Catalog and/or BLDB (21 to WT, 12 to ESBL and 4 to BLI resistant). Phylogenetic and comparative genomic analyses support that (i) SHV-1 (encoded by bla SHV-1 ) is the ancestral chromosomal variant, (ii) ESBL- and BLI-resistant variants have evolved multiple times through parallel substitution mutations, (iii) ESBL variants are mostly mobilized to plasmids and (iv) BLI-resistant variants mostly result from mutations in chromosomal bla SHV . We used matched genome-phenotype data from the KlebNET-GSP AMR Genotype-Phenotype Group to identify 3999 K . pneumoniae isolates carrying one or more bla SHV alleles but no other acquired β-lactamases to assess genotype-phenotype relationships for bla SHV . This collection includes human, animal and environmental isolates collected between 2001 and 2021 from 24 countries. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, whilst most omega-loop substitutions do not. Our data also provide support for the WT assignment of 67 protein variants, including 8 that were noted in public databases as ESBL. These eight variants were reclassified as WT because they lack ESBL-associated mutations, and our phenotype data support susceptibility to third-generation cephalosporins (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164 and SHV-187). The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotyping K. pneumoniae ), whereby known and novel bla SHV alleles are classified based on causative mutations. Kleborate v2.4.1 was updated to include ten novel protein variants from the KlebNET-GSP dataset and all alleles in public databases as of November 2023. This study demonstrates the power of sharing AMR phenotypes alongside genome data to improve the understanding of resistance mechanisms.

Epizootic haemorrhagic disease (EHD), caused by the EHD virus (EHDV), is a vector-borne viral disease transmitted through Culicoides biting midges. EHDV comprises seven serotypes (1, 2, and 4-8), with EHDV-8 having recently emerged and spread in Europe over the last two years. Such event has raised concerns about the significant threat posed by EHDV-8 to livestock industry. In this study, an inactivated vaccine against EHDV-8 (vEHDV8-IZSAM) was developed. Safety and efficacy of the vaccine were evaluated in calves through clinical, serological, and virological monitoring following experimental challenge. The vaccine was proven safe, with only transient fever and localized reactions observed in a few animals, consistent with adjuvanted vaccine side effects. vEHDV8-IZSAM elicited a robust humoral response, as evidenced by the presence of neutralizing antibodies. After challenge with a virulent isolate, viraemia and clinical signs were evidenced in control animals but in none of the vaccinated animals. This study highlights the potential of vEHDV8-IZSAM as a safe and highly effective vaccine against EHDV-8 in cattle. It offers protection from clinical disease and effectively prevents viraemia. With the recent spread of EHDV-8 in European livestock, the use of an inactivated vaccine could be key in protecting animals from clinical disease and thus to mitigate the economic impact of the disease. Further investigations are warranted to assess the duration of the induced immunity and the applicability of this vaccine in real-world settings. Accordingly, joint efforts between public veterinary institutions and pharmaceutical companies are recommended to scale up vaccine production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the IZSAM., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Despite the general classification of L. monocytogenes strains as equally virulent by global safety authorities, molecular epidemiology reveals diverse subtypes in food, processing environments, and clinical cases. This study focuses on a highly virulent strain associated with a listeriosis outbreak in Italy in 2022, providing insights through comprehensive foodomics approaches, with a specific emphasis on quantitative proteomics. In particular, the ST155 strain of L. monocytogenes strain was subjected in vitro to growth stress conditions (NaCl 2.4 %, pH 6.2, T 12 °C), mimicking the conditions present in the frankfurter, its original source. Then, the protein expression patterns were compared with those obtained in optimal growth conditions. Through quantitative proteomic analysis and bioinformatic assessment, different proteins associated with virulence during the exponential growth phase were identified. This study unveils unique proteins specific to each environment, providing insights into how L. monocytogenes adapts to conditions that are similar to those encountered in frankfurters. This investigation contributes valuable insights into the adaptive strategies of L. monocytogenes under stressful conditions, with implications for enhancing food safety practices., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Atrial fibrillation (AF) represents the most common supraventricular arrhythmia, with a prevalence of 1-3 % in the world population. Growing evidences show that AF plays an important role as a risk factor for the development of cognitive impairment (CoI) and dementia, depression and functional limitation. The purpose of the study is to evaluate, in a large cohort of elderly hospitalized patients with nonvalvular AF (NVAF) on direct oral anticoagulants (DOACs) therapy, the prevalence of CoI, depression, and functional limitation, and to assess the different variables that may be detrimental or protective on the risk of CoI or functional limitation. 1004 elderly patients were enrolled, 384 men and 620 women, with a mean age of 84±7.1 years. The two groups were comparable for the main study variables, except for age, prevalence of hypertension and CKD, which were higher in women, while ischemic heart disease was higher in men. In addition, the two groups differed in the CHA 2 DS 2 VASc score 5.3 ± 1.3 vs 4.2 ± 1.4 pts (p < 0.0001) and HAS-BLED score 2.5 ± 0.7 vs 2.3 ± 0.8 pts (p = 0.009) that were significantly higher in women. Our study revealed that in a cohort of elderly patients hospitalized with AF taking DOACs, CoI and disability are widely represented, and female sex increases the risk of being affected by CoI by about 3-fold, while improvement of functional limitations reduce this risk by about 15 %. In addition, CoI and depressive symptoms increase the risk of functional impairment about 2-fold and 28 % respectively, while antihypertensive and anti-diabetic therapy reduce this risk., Competing Interests: Declaration of competing interest The authors declare they have no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Purpose: Brucella canis is pathogenic for dogs and humans. Serological diagnosis is a cost-effective approach for disease surveillance, but a major drawback of current serological tests is the cross-reactivity with other bacteria that results in false positive reactions. Development of indirect tests with improved sensitivity and specificity that use selected B. canis proteins instead of the whole antigen remain a priority., Experimental Design: A western blotting assay was developed to define the serum antibody patterns associated to infection using a panel of positive and negative dog sera. B. canis positive sera recognized immunogenic bands ranging from 7 to 30 kDa that were then submitted to ESI-LC-MS/MS and analyzed by bioinformatics tools., Results: A total of 398 B. canis proteins were identified. Bioinformatics tools identified 16 non cytoplasmic immunogenic proteins predicted as non-homologous with the most important Brucella cross-reactive bacteria and nine B. canis proteins non-homologous to B. ovis; among the latter, one resulted non-homologous to B. melitensis. Data are available via ProteomeXchange with identifier PXD042682., Conclusions and Clinical Relevance: The western blotting test developed was able to distinguish between infected and non-infected animals and may serve as a confirmatory test for the serological diagnosis of B. canis. The mass spectrometry and in silico results lead to the identification of specific candidate antigens that pave the way for the development of more accurate indirect diagnostic tests., (© 2023 The Authors. Proteomics - Clinical Applications published by Wiley-VCH GmbH.)

Preventing L. monocytogenes infection is crucial for food safety, considering its widespread presence in the environment and its association with contaminated RTE foods. The pathogen's ability to persist under adverse conditions, for example, in food processing facilities, is linked to virulence and resistance mechanisms, including biofilm formation. In this study, the protein expression patterns of two L. monocytogenes 1/2a strains, grown under environmental stressors (mild acidic pH, thermal abuse, and high concentration of NaCl), were investigated. Protein identification and prediction were performed by nLC-ESI-MS/MS and nine different bioinformatic software programs, respectively. Gene enrichment analysis was carried out by STRING v11.05. A total of 1,215 proteins were identified, of which 335 were non-cytosolic proteins and 265 were immunogenic proteins. Proteomic analysis revealed differences in protein expression between L. monocytogenes strains in stressful conditions. The two strains exhibited unique protein expression profiles linked to stress response, virulence, and pathogenesis. Studying the proteomic profiles of such microorganisms provides information about adaptation and potential treatments, highlighting their genetic diversity and demonstrating the utility of bioinformatics and proteomics for a broader analysis of pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 D'Onofrio, Schirone, Krasteva, Tittarelli, Iannetti, Pomilio, Torresi, Paparella, D'Alterio and Luciani.)

Background: Genomic data-based machine learning tools are promising for real-time surveillance activities performing source attribution of foodborne bacteria such as Listeria monocytogenes. Given the heterogeneity of machine learning practices, our aim was to identify those influencing the source prediction performance of the usual holdout method combined with the repeated k-fold cross-validation method., Methods: A large collection of 1 100 L. monocytogenes genomes with known sources was built according to several genomic metrics to ensure authenticity and completeness of genomic profiles. Based on these genomic profiles (i.e. 7-locus alleles, core alleles, accessory genes, core SNPs and pan kmers), we developed a versatile workflow assessing prediction performance of different combinations of training dataset splitting (i.e. 50, 60, 70, 80 and 90%), data preprocessing (i.e. with or without near-zero variance removal), and learning models (i.e. BLR, ERT, RF, SGB, SVM and XGB). The performance metrics included accuracy, Cohen's kappa, F1-score, area under the curves from receiver operating characteristic curve, precision recall curve or precision recall gain curve, and execution time., Results: The testing average accuracies from accessory genes and pan kmers were significantly higher than accuracies from core alleles or SNPs. While the accuracies from 70 and 80% of training dataset splitting were not significantly different, those from 80% were significantly higher than the other tested proportions. The near-zero variance removal did not allow to produce results for 7-locus alleles, did not impact significantly the accuracy for core alleles, accessory genes and pan kmers, and decreased significantly accuracy for core SNPs. The SVM and XGB models did not present significant differences in accuracy between each other and reached significantly higher accuracies than BLR, SGB, ERT and RF, in this order of magnitude. However, the SVM model required more computing power than the XGB model, especially for high amount of descriptors such like core SNPs and pan kmers., Conclusions: In addition to recommendations about machine learning practices for L. monocytogenes source attribution based on genomic data, the present study also provides a freely available workflow to solve other balanced or unbalanced multiclass phenotypes from binary and categorical genomic profiles of other microorganisms without source code modifications., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The contamination of ready to eat foods (RTE) products due to Listeria monocytogenes could compromise the products safety becoming a great risk for the consumers. The high presence of L. monocytogenes in RTE products has been described worldwide, but few data are available about these products from African countries. The aims of this study were to report the presence of L. monocytogenes in Zambian RTE products, providing genomic characterization and data on similarity with African circulating strains using whole genome sequencing (WGS). A total of 304 RTE products, produced by different Zambian manufacturers, were purchased at retail, from major supermarkets located in Lusaka, Zambia, comprising 130 dairy and 174 meat products. L. monocytogenes was detected only in 18 (10.3%) RTE meat products of the 174 samples tested. The MLST analysis grouped the 18 L. monocytogenes isolates in 7 clonal complexes (CCs): CC1 ( n  = 5), CC2 ( n  = 4), CC9 ( n  = 4), CC5 ( n  = 2), CC121 ( n  = 1), CC155 ( n  = 1), and CC3 ( n  = 1). According to the cgMLST results, several clusters were detected, in particular belonging to hyper-virulent clones CC1 and CC2. Regarding the virulence factors, a complete L. monocytogenes Pathogenicity Island 3 (LIPI-3) was present both in the CC1 and CC3, in addition to LIPI-1. Several resistance genes and mobile genetic elements were detected, including Stress Islands, the bcrABC cassette and Tn6188 &#95;qac transposon, plasmids and intact prophages. Despite being a first preliminary work with a limited number of samples and isolates, this study helped to increase existing knowledge on contaminated RTE products in Zambia, confirming the presence of hyper-virulent L. monocytogenes CCs, which could play an important role in human diseases, posing a public health concern for consumers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Centorotola, Ziba, Cornacchia, Chiaverini, Torresi, Guidi, Cammà, Bowa, Mtonga, Magambwa, D’Alterio, Scacchia, Pomilio and Muuka.)

Brucella RB51 is a live modified vaccine. Its use in water buffalo has been proposed using a vaccination protocol different to that used for cattle, but knowledge of the long-term effects of RB51 vaccination in this species remains incomplete. The aim of the study was to evaluate the safety and kinetics of antibody responses in water buffaloes vaccinated according to the protocol described for the bovine species in the WOAH Manual, modified with the use of a triple dose. Water buffaloes were vaccinated with the vaccine RB51. A booster vaccination was administered at 12 months of age. When turning 23-25 months old, female animals were induced to pregnancy. RB51-specific antibodies were detected and quantified using a CFT based on the RB51 antigen. Vaccinated animals showed a positive serological reaction following each vaccine injection, but titers and the duration of the antibody differed among animals. For 36 weeks after booster vaccination, the comparison of CFT values between vaccinated and control groups remained constantly significant. Afterwards, antibody titers decreased. No relevant changes in antibody response were recorded during pregnancy or lactation. In conclusion, results indicated that the vaccination schedule applied is safe and allows for vaccinated and unvaccinated controls to be discriminated between for up to 8 months after booster vaccination.

In Europe, very few studies are available regarding the diversity of Listeria monocytogenes (L. monocytogenes) clonal complexes (CCs) and sequence types (ST) in poultry and on the related typing of isolates using whole genome sequencing (WGS). In this study, we used a WGS approach to type 122 L. monocytogenes strains isolated from chicken neck skin samples collected in two different slaughterhouses of an integrated Italian poultry company. The studied strains were classified into five CCs: CC1-ST1 (21.3%), CC6-ST6 (22.9%), CC9-ST9 (44.2%), CC121-ST121 (10.6%) and CC193-ST193 (0.8%). CC1 and CC6 strains presented a virulence gene profile composed of 60 virulence genes and including the Listeria Pathogenicity Island 3, aut&#95;IVb , gltA and gltB . According to cgMLST and SNPs analysis, long-term persistent clusters belonging to CC1 and CC6 were found in one of the two slaughterhouses. The reasons mediating the persistence of these CCs (up to 20 months) remain to be elucidated, and may involve the presence and the expression of stress response and environmental adaptation genes including heavy metals resistance genes ( cadAC, arsBC, CsoR-copA-copZ ), multidrug efflux pumps ( mrpABCEF, EmrB, mepA, bmrA, bmr3, norm ), cold-shock tolerance ( cspD ) and biofilm-formation determinants ( lmo0673, lmo2504, luxS, recO ). These findings indicated a serious risk of poultry finished products contamination with hypervirulent L. monocytogenes clones and raised concern for the consumer health. In addition to the AMR genes norB, mprF, lin and fosX , ubiquitous in L. monocytogenes strains, we also identified parC for quinolones, msrA for macrolides and tetA for tetracyclines. Although the phenotypical expression of these AMR genes was not tested, none of them is known to confer resistance to the primary antibiotics used to treat listeriosis The obtained results increase the data on the L. monocytogenes clones circulating in Italy and in particular in the poultry chain.

Listeria monocytogenes is a foodborne pathogen that is ubiquitous and largely distributed in food manufacturing environments. It is responsible for listeriosis, a disease that can lead to significant morbidity and fatality in immunocompromised patients, pregnant women, and newborns. Few reports have been published about proteome adaptation when L . monocytogenes is cultivated in stress conditions. In this study, we applied one-dimensional electrophoresis and 2D-PAGE combined with tandem mass spectrometry to evaluate proteome profiling in the following conditions: mild acid, low temperature, and high NaCl concentration. The total proteome was analyzed, also considering the case of normal growth-supporting conditions. A total of 1,160 proteins were identified and those related to pathogenesis and stress response pathways were analyzed. The proteins involved in the expression of virulent pathways when L . monocytogenes ST7 strain was grown under different stress conditions were described. Certain proteins, particularly those involved in the pathogenesis pathway, such as Listeriolysin regulatory protein and Internalin A, were only found when the strain was grown under specific stress conditions. Studying how L. monocytogenes adapts to stress can help to control its growth in food, reducing the risk for consumers.

West Nile virus (WNV) is an important zoonotic Flavivirus responsible for mild fever to severe neurological disease in humans and horses. Despite the occurrence of major previous outbreaks in Namibia and the likelihood of the current endemicity of the virus, only limited investigations and monitoring activities of WNV have been performed in the country. The use of animal sentinels is a valuable approach toward investigating the infection presence in an area and to predict the potential occurrence of human outbreaks. Serological investigations in dogs hold several advantages, considering their infection susceptibility, the ease of sample handling, and the evaluation of risk factors of pet owners that share the same habit with their pets. To evaluate the usefulness of such a sero-epidemiological investigation in Namibia, a broad serosurvey was performed in 2022 that included 426 archived domestic dog samples from eight Namibian regions. Although the ELISA prevalence, indicative of Flavivirus infection, was relatively high (16.43%; 95 CI: 13.10-20.39%), the virus neutralization test confirmed only a minority of cases, highlighting a prevalence of 2.82% (95 CI: 1.47-4.90%), significantly lower than in Namibian donkeys and reports from other countries. Variables that could explain the recorded differences remain to be explored, including animal exposure, variable vector presence, distribution, and feeding preferences. The study results suggest the limited usefulness of dogs as sentinels for WNV monitoring in Namibia.

The measure of hair cortisol concentration (HCC) is becoming an emerging approach to monitor mid-/long-term stress in animals, so it is more and more important to develop accurate and reliable methods. In the light of this, the aim of the present study was to compare mane HCCs of 47 horses with different managements, by means of an immunoassay (ELISA) and liquid chromatography coupled to hybrid high-resolution mass spectrometry (LC-HRMS/MS). After the washing step, the ground hair was extracted with methanol. The extract was evaporated and redissolved in two different aqueous solutions, depending on the detection technique. The methods were validated according to EMA guideline for bioanalytical method validation, in the range 2-50 pg mg -1 (ELISA) and 1-100 pg mg -1 (LC-HRMS/MS). Satisfactory quantitative performances were obtained for both of the approaches, but this latter demonstrated better precision. The detected concentrations in real samples were encompassing the range 1.3-8.8 pg mg -1 and 2.0-17.9 pg mg -1 by means of LC-HRMS/MS and ELISA, respectively. Overall, HCCs measured with ELISA technique were 1.6 times higher. The overestimation of immunoassay results might be caused by cross-reactivity phenomena of laboratory reagents and other structurally similar hormones present in the mane., (© 2022. The Author(s).)

A 15-year-old neutered male mixed breed Domestic Shorthair cat was presented for a rapidly growing, intraoral soft gingival mass on the left mandibular region. The neoplastic tissue consisted histologically of two distinct malignant cell populations: spindle cells arranged in bands and epithelioid cells arranged in cords. A few multinucleated giant cells were scattered among the neoplastic cells. Spindle cells and multinucleated giant cells strongly expressed vimentin while epithelial cells strongly expressed pancytokeratins. On the basis of the histological and immunohistochemical results, a diagnosis of oral carcinosarcoma was made. After 2 months, due to the extent of disease and poor prognosis, the cat was euthanized. Necropsy revealed a markedly enlarged, multilobulated white-pink neoplastic mass that had originated from the left side of the sublingual region and involved the coronoid process of the left mandibular bone. The cut surface of the enlarged left submandibular lymph node was glistening, whitish-tan in colour with a multinodular appearance, suggestive of metastasis and confirmed by histological examination. Oral carcinosarcoma is uncommonly recorded in humans and dogs and, to the best of our knowledge, this is the first reported case in a cat., Competing Interests: Conflict of Interest Statement The authors declared no potential conflicts of interest with respects to the research, authorship or publication of this article., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Horses have always been animals used for companionship, work, transportation, and performance purposes over the history of humanity; there are different ways of managing horses, but studies on how horse welfare is influenced by different activities and managements are scanty. Understanding how the management, the environment, and the different uses of horses can affect the level of stress and well-being is important not only for people associated with horses. Three groups of horses with different management, environments, and activities were selected: (1) stabled horses ridden frequently, (2) horses that perform public order service under the Italian state police, and (3) free-ranging horses. Cortisol analysis was carried out on horsehair samples using liquid chromatography coupled to hybrid orbitrap high-resolution mass spectrometry (LC-HRMS/MS), a laboratory technique used for the first time to quantify horsehair cortisol. The selection of horses to be included in the three groups was carried out by including only subjects with positive welfare assessment in accordance with the horse welfare assessment protocol (AWIN). These analyses demonstrated that the cortisol levels detected in the horsehair of free-ranging animals were significantly higher compared to those detected in stabled and working horses. These results may have been a consequence of complex environmental, managerial, and behavioral factors, which should be worth further investigation.

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.

From 24 December 2020 to 8 February 2021, 163 cases of SARS-CoV-2 Alpha variant of concern (VOC) were identified in Chieti province, Abruzzo region. Epidemiological data allowed the identification of 14 epi-clusters. With one exception, all the epi-clusters were linked to the town of Guardiagrele: 149 contacts formed the network, two-thirds of which were referred to the family/friends context. Real data were then used to estimate transmission parameters. According to our method, the calculated Re(t) was higher than 2 before the 12 December 2020. Similar values were obtained from other studies considering Alpha VOC. Italian sequence data were combined with a random subset of sequences obtained from the GISAID database. Genomic analysis showed close identity between the sequences from Guardiagrele, forming one distinct clade. This would suggest one or limited unspecified viral introductions from outside to Abruzzo region in early December 2020, which led to the diffusion of Alpha VOC in Guardiagrele and in neighbouring municipalities, with very limited inter-regional mixing., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Human listeriosis outbreaks are often associated with food products, which could be contaminated, at the same time, also by different clones of Listeria monocytogenes. This emphasize the need to type more than one L.monocytogenes isolate found in a single food or environmental sample. The purpose of the present study was to evaluate the presence of different L.monocytogenes strains in food and food production environment in order to understand if there is need to type more isolates from the same sample in case of presence of L.monocytogenes. Between 2011 and 2015, at the Italian National Reference Laboratory for L.monocytogenes, for each positive sample, from two to twenty-three isolates of L.monocytogenes were collected. All the isolates were characterized by conventional serotyping and pulsed field gel electrophoresis (PFGE). Moreover, isolates from the same sample, having indistinguishable PFGE profile, were subjected to whole genome sequencing in order to perform core genome Multi Locus Sequence Typing (cgMLST). Within each sample, more than one serotype and one pulsotype were found in 11.9% and 27.5%, respectively. For indistinguishable PFGE patterns the cgMLST analysis showed 96.2% of concordance demonstrating the added value of new sequencing technologies. This study has demonstrated the need to select and type more than one L.monocytogenes colony in one food or food environmental sample to detect the diversity of L.monocytogenes strains and facilitate downstream investigations and effective source attribution in foodborne outbreak inquiry., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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&#95;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.

West Nile virus (WNV) and Usutu virus (USUV) are the two most widespread mosquito-borne flaviviruses in Europe causing severe neuroinvasive disease in humans. Here, following standardization of the murine model with wild type (wt) viruses, we engineered WNV and USUV genome by reverse genetics. A recombinant virus carrying the 5' UTR of WNV within the USUV genome backbone (r-USUV 5'-UTR WNV ) was rescued; when administered to mice this virus did not cause signs or disease as wt USUV suggesting that 5' UTR of a marked neurotropic parental WNV was not per se a virulence factor. Interestingly, a chimeric virus carrying the envelope (E) protein of USUV in the WNV genome backbone (r-WNV E-USUV ) showed an attenuated profile in mice compared to wt WNV but significantly more virulent than wt USUV. Moreover, except when tested against serum samples originating from a live WNV infection, r-WNV E-USUV showed an identical antigenic profile to wt USUV confirming that E is also the major immunodominant protein of USUV., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Several lineages of SARS-CoV-2 are currently circulating worldwide. During SARS-CoV-2 diagnostic activities performed in Abruzzo region (central Italy) several strains belonging to the B.1.177.75 lineage tested negative for the N gene but positive for the ORF1ab and S genes (+/+/- pattern) by the TaqPath COVID-19 CE-IVD RT-PCR Kit manufactured by Thermofisher. By sequencing, a unique mutation, synonymous 28948C > T, was found in the N-negative B.1.177.75 strains. Although we do not have any knowledge upon the nucleotide sequences of the primers and probe adopted by this kit, it is likely that N gene dropout only occurs when 28948C > T is coupled with 28932C > T, this latter present, in turn, in all B.1.177.75 sequences available on public databases. Furthermore, epidemiological analysis was also performed. The majority of the N-negative B.1.177.75 cases belonged to two clusters apparently unrelated to each other and both clusters involved young people. However, the phylogeny for sequences containing the +/+/- pattern strongly supports a genetic connection and one common source for both clusters. Though, genetic comparison suggests a connection rather than indicating the independent emergence of the same mutation in two apparently unrelated clusters. This study highlights once more the importance of sharing genomic data to link apparently unrelated epidemiological clusters and to, remarkably, update molecular tests.

Brucella canis has been isolated for the first time in Italy in a commercial breeding kennel. It was diagnosed after a deep investigation related to the onset of reproductive disorders. Animals were tested with direct and indirect techniques. The agent was first detected in two Chihuahua aborted foetuses by direct culture. Further, it was also isolated from blood samples of dogs hosted in the kennel, which also showed reaction to conventional serological tests (microplate serum agglutination test). The isolates were identified as B. canis by standard microbiological methods and a Bruce‑ladder multiplex PCR. To investigate the genomic diversity, whole genome sequencing was used, applying the core genome Multilocus Sequence Typing (cgMLST ). In a first round of serological testing performed on 598 animals, 269 (46.1%) tested positive. In the second round of laboratory testing carried out 4‑5 weeks apart, the number of serologically positive dogs was 241 out of 683 tested (35.3%), while the number of dogs positive to isolation was 68 out of 683 tested (10.0%). The PCR showed a lack of sensitivity when compared to direct isolation. The epidemiological investigation did not identify the source of the infection, given the time elapsed from the onset of abortions to the definitive diagnosis of B. canis infection in the kennel. The genomic analyses featured the strains as ST21 and, according to the cgMLST, revealed the presence of a tight cluster with a maximum diversity of four allelic differences. The observed limited genomic variation, largely within the known outbreak cut‑offs, suggests that the outbreak herein described was likely caused by a single introduction. Moreover, in a broader scale comparison using the public available genomes, we found that the closest genome, isolated in China, differed by more than 50 alleles making not possible to find out the likely origin of the outbreak. The lack of updated data on B. canis genome sequences in the public databases, together with the limited information retrieved from the epidemiological investigations on the outbreak, hampered identification of the source of B. canis infection.

West Nile Virus (WNV) is a mosquito-borne virus enzootically maintained in birds. However, it can incidentally infect other species, leading to sometimes severe clinical consequences like in horses and especially human beings. Despite the topic relevance, the presence and distribution of WNV are currently unknown in Namibia. Several countries implement surveillance systems based on virus detection in birds, mosquitoes, and vertebrate species including horses. The present study aimed to fill this knowledge gap by serologically evaluating WNV exposure in Namibian donkeys, whose population is remarkably bigger than the horse one. Forty-seven out of 260 sampled animals showed neutralizing antibodies against WNV (18.07% [95% CI = 13.59-23.30%]), demonstrating its circulation in all country territory, although, with apparent regional differences. On the contrary, no association with animal age or sex could be identified. The present study demonstrates the widespread presence of WNV in Namibia as well as the practical utility and effectiveness of donkeys as sentinels for infection surveillance. Due to clinical relevance, vaccination campaigns should be considered for horses of high economic or genetic value. Additionally, the burden of WNV infection on human health should be carefully evaluated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Molini, Franzo, Nel, Khaiseb, Ntahonshikira, Chiwome, Baines, Madzingira, Monaco, Savini and D'Alterio.)

From 16 March to 15 December 2020, 132,357 naso-pharyngeal/oropharyngeal swabs were collected in the province of Teramo, Abruzzo Region, Italy, and tested for the presence of SARS-CoV-2 genomic RNA by a commercially available molecular assay. A total of 12,880 swabs resulted positive. For 8212 positive patients (4.150 women and 4.062 men) the median age was statistically different between women (median: 49.55 ± 23.9 of SD) and men (median: 48.35 ± 23.5 of SD) while no differences were found in the comparison between the cycle threshold for the N protein-encoding gene (C T N) median values and gender. Differences were observed in the C T N gene median values of swabs collected from March to September as well as in the pairwise comparison between September and October and between November and December. The C T N gene median values observed in specific periods characterizing the SARS-CoV-2 epidemic in 2020 were also compared with the incidence of COVID-19 cases; a strong inverse correlation was highlighted (Pearson correlation coefficient = -0.978). Our findings confirm the usefulness of the C T N values as an indirect detection parameter to monitor viral loads in the population.

Pecorino is a typical Italian cheese, mostly produced in central and southern Italy regions using ewe raw milk and following traditional procedures. The use of raw milk constitutes a risk linked to the potential survival or multiplication of pathogenic microorganisms, as Shiga toxin-producing Escherichia coli (STEC). The aim of this study was to compare different Italian traditional Pecorino production methods to determine if there were any phases that could influence the Escherichia coli O157 survival rate, but also if they could negatively influence lactic acid bacteria survival rate, during the phases of production and ripening. Therefore batches of Pecorino cheese were prepared using different production methods, representing the real and typical cheese production in southern and central Italy regions: 1) heating the milk at 37 °C for about 40 min before curding, 2) heating the milk at 60 °C (thermization) for 13 min, so that the alkaline phosphatase reaction is still positive before curding, 3) cooking curd at 41 °C and 4) at 45 °C, both for 5 min. Our results demonstrated that traditional milk treatments different from pasteurization can help but do not eliminate serious microbiological treats, as E. coli O157, especially if the raw milk is heavily contaminated. The heat treatment at 60 °C applied to raw milk was able to decrease the concentration of E. coli O157 of 1.7 log 10 CFU/ml and, according to the inactivation slope, it would be further reduced prolonging the heating treatment. The results obtained also showed that, during the Pecorino cheese ripening, E. coli O157 was always enumerable for 60 days, remaining detectable after 90 days of ripening., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In Europe wildlife animals such as the red fox ( Vulpes vulpes ) are considered the main reservoir for Angiostrongylus vasorum as well as a potential threat for domestic dog infection. Though this parasite is endemic in fox populations, data on A. vasorum infection in wolves ( Canis lupus italicus ) are still scant, having only recently been described in Northwestern Spain, in Italy, in Croatia and in Slovakia. Based on the rising number of cases of canine lungworm infection in Central Italy (Abruzzo region), the aim of the present study was to investigate the infection by A. vasorum in fox and wolf populations sharing the same geographical area of dogs. From October 2008 to November 2019, A. vasorum specimens were collected, through routine post-mortem examination, from 56 carcasses (44 foxes and 12 wolves). Adult parasites were searched for in the right side of the heart and in pulmonary artery of all carcasses. First stage of larvae (L1) was searched in faeces using the Baermann technique and in lungs by tissue impressions. Overall, 230 adult specimens were collected and identified on a morphological basis. To confirm the morphological identification, 4 adult specimens (n = 3 from fox, n = 1 from wolf) were molecularly identified as A. vasorum by amplification of partial fragment of nuclear 18S rRNA (~1700 bp) genes. The anatomo-pathological and parasitological examinations indicated the presence of A. vasorum in 33 foxes (75%) and in 8 wolves (66.7%). The level of prevalence of infested wolves was higher than the previous one reported in other European countries. Interestingly, the prevalence of infection in foxes herein recorded was higher than that described in dogs (8.9%) living in the same geographical area. This result may confirm the hypothesis that the spread of canine angiostrongylosis is linked to fox populations infection., Competing Interests: None., (© 2021 The Authors.)

The ultimate goal of any disaster response, or a natural or a man‑made event, is to get the best outcome for the highest number of people. From a veterinary point of view, the best outcome includes either the protection of animals (conventional and unconventional pets) or the safeguarding the wholesomeness of food supplies in the "One Health" perspective. The evolution of the Italian veterinary role in disaster management has changed across the last 35 years and has grown with the awareness that animals and human beings share the same vulnerability to disasters. The University of Teramo, following its experiences in different disaster scenarios, proposes a veterinary presidium to support Public Authority in responding to catastrophic events in the Italian context, in order to rescue small, large and unconventional animals. The proposed veterinary presidium is made up of 3 skilled people certified to react to different population needs. Indeed we propose different teams to rescue small, large or non‑conventional animal, trained to work together in a stress situation and under coordination of the Civil Protection Function 2. This presidium with its 3 different skilled teams under the supervision of the advanced veterinary medical center (AVMC) and by reporting to it will provide the best competences based on the needs of the population and the authorities, in view of the "One Health" perspective.

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).

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.

There is strong evidence that severe acute respiratory syndrome 2 virus (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, originated from an animal reservoir. However, the exact mechanisms of emergence, the host species involved, and the risk to domestic and agricultural animals are largely unknown. Some domestic animal species, including cats, ferrets, and minks, have been demonstrated to be susceptible to SARS-CoV-2 infection, while others, such as pigs and chickens, are not. Importantly, the susceptibility of ruminants to SARS-CoV-2 is unknown, even though they often live in close proximity to humans. We investigated the replication and tissue tropism of two different SARS-CoV-2 isolates in the respiratory tract of three farm animal species - cattle, sheep, and pigs - using respiratory ex vivo organ cultures (EVOCs). We demonstrate that the respiratory tissues of cattle and sheep, but not of pigs, sustain viral replication in vitro of both isolates and that SARS-CoV-2 is associated to ACE2-expressing cells of the respiratory tract of both ruminant species. Intriguingly, a SARS-CoV-2 isolate containing an amino acid substitution at site 614 of the spike protein (mutation D614G) replicated at higher magnitude in ex vivo tissues of both ruminant species, supporting previous results obtained using human cells. These results suggest that additional in vivo experiments involving several ruminant species are warranted to determine their potential role in the epidemiology of this virus., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Orchitis and epididymo-orchitis are inflammatory lesions of the testicle. We herein describe a case of monolateral chronic orchitis which occurred in a Tiro Pesante Rapido (TPR) stallion, born in 2002, with a history of good fertility. The stallion was healthy and asymptomatic although the left testis was found to be smaller as compared with the right one and was hard in consistency. Histopathology examination revealed tubular atrophy and parenchymal sclerosis. Scattered foci of calcification and chronic inflammation, the latter dominated by macrophages and lymphocytes, were also observed. Although lesions were clearly present, the semen was demonstraed to be of good quality. This study highlights the need for periodic clinical and ultrasound evaluation of stallions, in order to preserve their reproductive performance.

The aim of this study was to evaluate the performance of "Micro Biological Survey - MBS Test" in the enumeration of bacterial load in cow raw milk. The MBS test is based on a colorimetric method recently developed and patented by "Roma Tre" University, Italy. The evaluation of the performance of the MBS method was carried out by comparison with plate count at 30°C (gold standard) and flow cytometry. Thirteen independent set of experiments were performed analyzing a total of 104 samples of cow raw milk with the selected methods. Results obtained using the MBS method are comparable with those obtained with the plate count method at 30°C (CFU/mL) and flow cytometry technology; in particular, the results obtained with the MBS method are very close to plate count's at 30°C. On the other hand, there are statistically significant differences between these two methods' and flow cytometry technology's results that could be due to the different experimental conditions., Competing Interests: Conflict of interests: The authors declare no potential conflict of interests., (©Copyright: the Author(s).)

By late March 2020, Villa Caldari, a small village of the municipality of Ortona (Abruzzo region), was registering an incidence rate of COVID-19 cases ten times greater than the overall municipality and was declared a hotspot area. Twenty-two days later, epidemiological investigation and sampling were performed, to evaluate SARS-CoV-2 circulation and the presence of SARS-CoV-2 antibodies. Overall, 681 nasopharyngeal swabs and 667 blood samples were collected. Only one resident of the village resulted in being positive for RNA viral shedding, while 73 were positive for SARS-CoV-2 antibodies. The overall seroprevalence was 10.9%. The difference between the seroprevalence of infection in asymptomatic and symptomatic individuals was significant (χ 2 = 14.50 p -value = 0.0001). Amongst the residents positive for antibodies, fatigue and/or muscle pain, fever and anosmia were the most experienced symptoms, whose most frequent onset was observed during the first two weeks of March. Familial and habit-related clusters were highlighted. Nevertheless, the investigations showed a low SARS-CoV-2 circulation in the village at the time of the sampling, demonstrating virus transmission could be limited when strict emergency measures are followed. Given the favorable results, the emergency measures were then lifted.