Virus-Mediated Metalloproteinase 1 Induction Revealed by Transcriptome Profiling of Bovine Herpesvirus 4-Infected Bovine Endometrial Stromal Cells.

Viral infections can cause genital tract disorders (including abortion) in cows, and bovine herpesvirus 4 (BoHV-4) is often present in endometritis-affected animals. A major problem with cattle uterine viral infections in general, and BoHV-4 in particular, is our limited understanding of the pathogenic role(s) that these infections play in the endometrium. A similar lack of knowledge holds for the molecular mechanisms utilized, and the host cell pathways affected, by BoHV-4. To begin to fill these gaps, we set up optimized conditions for BoHV-4 infection of a pure population of bovine endometrial stromal cells (BESCs) to be used as source material for RNA sequencing-based transcriptome profiling. Many genes were found to be upregulated (417) or downregulated (181) after BoHV-4 infection. As revealed by enrichment functional analysis on differentially expressed genes, BoHV-4 infection affects various pathways related to cell proliferation and cell surface integrity, at least three of which were centered on upregulation of matrix metalloproteinase 1 (MMP1) and interleukin 8 (IL8). This was confirmed by reverse transcription PCR, real-time PCR, Western-immunoblot analysis, and a luciferase assay with a bovine MMP1-specific promoter reporter construct. Further, it was found that MMP1 transcription was upregulated by the BoHV-4 transactivator IE2/RTA, leading to abnormally high metalloproteinase tissue levels, potentially leading to defective endometrium healing and unresolved inflammation. Based on these findings, we propose a new model for BoHV-4 action centered on IE2-mediated MMP1 upregulation and novel therapeutic interventions based on IFN gamma-mediated MMP1 downregulation.

BoHV-4-based vector delivering Ebola virus surface glycoprotein.

BACKGROUND: Ebola virus (EBOV) is a Category A pathogen that is a member of Filoviridae family that causes hemorrhagic fever in humans and non-human primates. Unpredictable and devastating outbreaks of disease have recently occurred in Africa and current immunoprophylaxis and therapies are limited. The main limitation of working with pathogens like EBOV is the need for costly containment. To potentiate further and wider opportunity for EBOV prophylactics and therapies development, innovative approaches are necessary. METHODS: In the present study, an antigen delivery platform based on a recombinant bovine herpesvirus 4 (BoHV-4), delivering a synthetic EBOV glycoprotein (GP) gene sequence, BoHV-4-syEBOVgD106ΔTK, was generated. RESULTS: EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106ΔTK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106ΔTK immunized goats produced high titers of anti-EBOV GP antibodies and conferred a long lasting (up to 6 months), detectable antibody response. Furthermore, no evidence of BoHV-4-syEBOVgD106ΔTK viremia and secondary localization was detected in any of the immunized animals. CONCLUSIONS: The BoHV-4-based vector approach described here, represents: an alternative antigen delivery system for vaccination and a proof of principle study for anti-EBOV antibodies generation in goats for potential immunotherapy applications.

BoHV-4 immediate early 1 gene is a dispensable gene and its product is not a bone marrow stromal cell antigen 2 counteracting factor.

BACKGROUND: Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus whose genome was cloned as Bacterial Artificial Chromosome (BAC) and exploited as a gene delivery vector for vaccine purposes. Although BoHV-4 genome has been completely sequenced and its open reading frames (ORFs) structurally defined in silico, most of them are not functionally characterized. In BoHV-4 genome two major immediate early genes (IE) are present, IE1 and IE2. IE2 is an essential gene because its removal from the viral genome renders the virus unable to replicate, whereas for IE1 no many functional information are available. RESULTS: In this work, IE1 contribution in initiating and maintaining BoHV-4 lytic replication was assessed generating a recombinant BoHV-4 genome lacking of IE1 gene, BoHV-4ΔIE1. In contrast to BoHV-4IE2 deleted mutant, BoHV-4ΔIE1 infectious replicating viral particles (IRVPs) could be reconstituted following viral DNA electroporation in permissive cells. However the titer of BoHV-4ΔIE1 IRVPs produced into the cell supernatant and BoHV-4ΔIE1 plaques size were reduced respect to BoHV-4 undeleted control. Further the impaired BoHV-4ΔIE1 IRVPs produced into the cell supernatant could be rescued by expressing IE1 gene product in trans, confirming the implication of IE1 in BoHV-4 lytic replication. Next, the possible role of BoHV-4IE1 as bone marrow stromal cell antigen 2 (BST-2) counteracting factor, as hypothesized by IE1 amino-terminal gene product homology with Kaposi Sarcoma Associated Herpesvirus (KSHV) K5, was excluded too. CONCLUSIONS: Although the real function of BoHV-4IE1 is still elusive, a new BoHV-4 genome gene locus as a target site for the insertion of foreign DNA and resulting in the attenuation of the virus has been revealed. These data can be considered of relevance to improve BoHV-4 gene delivery properties.

Intestinal Emphysema and Gut Bacterial Microbiota Composition.

Pneumatosis cystoides intestinalis, or intestinal emphysema, is a condition characterized by the presence of multiple cystic structures within the gut wall and on the serosal surface of the intestine. Intestinal emphysema represents an accidental finding in swine, although it can be clinically relevant in humans. Its etiology is unknown, and many theories have been proposed. Among them, a bacterial etiology is considered the most likely. Therefore, in this study, the V3-V4 region of the 16S rRNA gene was sequenced from 19 swine ileal tracts, 12 with intestinal emphysema and 7 without lesions, to detect a possible bacterial agent. In parallel, prevalence was estimated. Escherichia-Shigella (13.15%), Clostridium_sensu_stricto_1; s__uncultured_bacterium (7.09%), and Fusobacterium; s_uncultured bacterium (6.60%) were the most abundant species identified. No statistically relevant differences were observed between the pathological and physiological groups. Prevalence ranged from 1.25 to 5.12% depending on the batch. Our results suggest that the gut wall bacterial microbiota greatly match the normal gut microbiota, and that the etiological agent of intestinal emphysema may be (1) undetectable due to the chronicity of the lesions, (2) not considered statistically relevant in comparing the two groups (p < 0.05) and likewise in causing lesions, and (3) undetectable due to contamination. Regarding prevalence, the condition is moderately frequent.

Re-determination of the primary shelf-life of food products: what are the guarantees for the consumer?

Except in rare cases, the determination of the shelf-life of food products is left up to the food business operator. The extension of this period, which for years has been the subject of debate among the various actors in the food chain, has become a topic of fundamental importance also following the recent economic/financial, environmental, and health crises, which have had an inevitable impact on consumption and food waste. While there is no requirement to indicate durability for some categories of food products, for example, those not directly intended for consumers, this debate has raised questions and perplexities about the potential re-evaluation of the origin conditions established by the manufacturer, particularly when it comes to maintaining the guarantees for the consumer in terms of health and hygiene. In addition, the increasing consumer demand for accurate information has prompted the European authorities to request a public consultation on the actual understanding and perception of the mandatory terms on labels such as use by or date of minimum durability of a food, provided for by Article 9 of Regulation (EU) No. 1169/2011, often not correctly understood, which can assume great significance in the application of rules to reduce food waste. In this regard, it is useful to remember that the recent measures adopted by the European Union legislator, together with the case law of recent years, have led the judges of merit to comply with the principles and requirements of food safety laid down since 2002 in Regulation (EC) No. 178, thus paying greater attention to the analysis, assessment, and management of the risk of the entire production chain. The purpose of this work is to provide technical-legal elements to encourage a possible extension of the shelf-life of food products while ensuring the safety of consumers.

Condemnation of Porcine Carcasses: A Two-Year Long Survey in an Italian High-Throughput Slaughterhouse.

Ante-mortem and post-mortem inspection at slaughter are performed by Official Veterinarians and it is essential to identify alterations/lesions, which can make organs/carcasses unsuitable for human consumption. Obviously, carcass condemnation must be regarded as a highly undesirable event for the entire swine industry chain, as it represents the total failure of a long period of challenging work. Therefore, it seems valuable to estimate the prevalence and causes of carcasses condemnation, in order to preserve consumers' health and pig farming profitability. Bearing that in mind, the present study aimed at providing a reliable picture of the condemnation of porcine carcasses in Italy, with a special emphasis being placed upon pathological findings. Data were collected in a high-throughput abattoir located in northern Italy, where a total of 2,062,278 pigs were slaughtered during the period of study (2021-2022). Overall, 1362 pigs were considered unfit for slaughtering after ante-mortem inspection, mostly because of death during transportation or in resting pens, after their arrival at the abattoir. Moreover, 2007 carcasses were condemned after post-mortem inspection. The most common causes of condemnation were severe and diffuse peritonitis, disseminated abscesses, jaundice, and erysipelas. In our opinion, the present survey may provide useful and updated information about the condemnation of pig carcasses in Italy. At the same time, it highlights the need to collect data in a more systematic and standardized way, thus making possible their comparison over time and among different geographic areas.

Scoring Enzootic Pneumonia-like Lesions in Slaughtered Pigs: Traditional vs. Artificial-Intelligence-Based Methods.

Artificial-intelligence-based methods are regularly used in the biomedical sciences, mainly in the field of diagnostic imaging. Recently, convolutional neural networks have been trained to score pleurisy and pneumonia in slaughtered pigs. The aim of this study is to further evaluate the performance of a convolutional neural network when compared with the gold standard (i.e., scores provided by a skilled operator along the slaughter chain through visual inspection and palpation). In total, 441 lungs (180 healthy and 261 diseased) are included in this study. Each lung was scored according to traditional methods, which represent the gold standard (Madec's and Christensen's grids). Moreover, the same lungs were photographed and thereafter scored by a trained convolutional neural network. Overall, the results reveal that the convolutional neural network is very specific (95.55%) and quite sensitive (85.05%), showing a rather high correlation when compared with the scores provided by a skilled veterinarian (Spearman's coefficient = 0.831, p < 0.01). In summary, this study suggests that convolutional neural networks could be effectively used at slaughterhouses and stimulates further investigation in this field of research.

Microbiological and Chemical Analysis of Food Collected Under Official Control in the Emilia-Romagna Region of Northern Italy, 2014-2019.

This study analyzed data from 6 years (2014-2019) of official controls in the Emilia-Romagna region (northern Italy) to investigate the frequencies of human pathogens and chemical hazards in foods during production and distribution. Campylobacter spp. was the most prevalent pathogen, isolated in 4.4% of the 1,078 food samples examined, followed by Salmonella spp. (2.8%), Shiga toxin-producing Escherichia coli (STEC) (1.9%), and Listeria monocytogenes (0.9%). Salmonella serotyping showed that the isolates belonged to the serotypes most commonly isolated from humans in Emilia-Romagna. These serotypes were as follows: S. Infantis (34.8%), mostly isolated from chicken, monophasic S. Typhimurium (1,4, [5],12:i:-) (12.6%), S. Bredeney (8.9%), and S. Derby (8.6%). No Clostridium botulinum, Yersinia spp., and Shigella spp. were isolated. No positivity was detected for hepatitis A virus, while 5.1% of samples taken in the production phase of the food chain were found to be contaminated with norovirus. The chemical analyses identified environmental contaminants within legal limits (heavy metals, 0.6% positive overall; mycotoxins, 0.4% positive overall), analytes subjected to monitoring (perfluoro-alkyl substances (PFASs), 6.2% positive overall; inorganic arsenic, no positives overall) and process contaminants and additives within legal limits (acrylamide, 9.6% positive overall; permitted or nonpermitted additives, 0.9% positive overall). Only one sample showed dioxins and polychlorinated biphenyls (PCBs) at levels higher than the legal limits. The monitoring by competent authorities (CA) of food contamination can generate useful data that can be used as a basis for estimating the exposure to different food contaminants over time and for evaluating the effects of control measures on the contamination of food.

Training Convolutional Neural Networks to Score Pneumonia in Slaughtered Pigs.

The slaughterhouse can act as a valid checkpoint to estimate the prevalence and the economic impact of diseases in farm animals. At present, scoring lesions is a challenging and time-consuming activity, which is carried out by veterinarians serving the slaughter chain. Over recent years, artificial intelligence(AI) has gained traction in many fields of research, including livestock production. In particular, AI-based methods appear able to solve highly repetitive tasks and to consistently analyze large amounts of data, such as those collected by veterinarians during postmortem inspection in high-throughput slaughterhouses. The present study aims to develop an AI-based method capable of recognizing and quantifying enzootic pneumonia-like lesions on digital images captured from slaughtered pigs under routine abattoir conditions. Overall, the data indicate that the AI-based method proposed herein could properly identify and score enzootic pneumonia-like lesions without interfering with the slaughter chain routine. According to European legislation, the application of such a method avoids the handling of carcasses and organs, decreasing the risk of microbial contamination, and could provide further alternatives in the field of food hygiene.

Official controls on food safety: Competent Authority measures.

Since December 14th, 2019, Regulation (EC) No 882/2004 has been replaced by Regulation (EU) 2017/625, which sets the activity of the official control on food safety, extending the scope of the previous regulation. The broader scope of the new regulation aims to ensure compliance across the European Member States in the Union in the fields of food, feed, animal health and welfare, plant health and plant protection products. The administrative measures that the Competent Authorities adopt following the finding of a non-compliance regarding food hygiene, should be take into account not only the risk assessment, but a series of criteria dictated by both European and national legislation and comply with the generals principles governing administrative action. The aim of this study is to conduct a legal analysis of: (i) the provisions set out in Regulation (EU) 2017/625 concerning executive actions in the case of noncompliance, and (ii) the criteria used to assess the appropriate remedial measures. The study was completed by analysing recent legal cases on food safety, which in many cases have censure the work of the Local Competent Authority, and has brought to light a distorted and uneven application of the legislation on food safety, especially in cases in which drastic measures had been adopted, such as the closure of the productive activities, which has a heavy impact on the food business operators in the food sector concerned. In addition to an incorrect application of the specific health legislation, there is also a violation of the principle of proportionality, of community origin. Indeed, the principle of proportionality, an essential factor in administrative review, which has been used as a leading criterion in the adoption of administrative measures by of the Health Authorities of the Competent Authority.

Farm products' direct sale in accordance with national and EC Regulations.

Primary production has always been considered the weak link in the entire food production chain (from farm to fork) and, due also to the grave health and food emergencies that have taken place over the years (BSE, dioxin, avian flu etc.), greater attention has been focused on the production stage, together with the need to regain the consumers' faith. To preserve and support small farms in a local setting and, consistent with the aims of flexibility and respecting the main requisites contained in the EC Regulations (No. 852/2004; No. 853/2004) (European Commission, 2004; 2004a), production is allowed for tastingadministration on the premises and the processing and sales of agricultural products produced exclusively on the farm, such as: fresh meat from poultry and rabbits and small farmed wild animals; processed meats obtained from animals raised on the farm and from hunting; fishing and aquaculture products; raw milk for direct human consumption and dairy products; eggs, honey, fruit and vegetables, woodland products; jams and preserved fruit, flours, vegetable preserves, wild above ground and underground mushrooms; dried fruits, fruit juices, cereals, syrups; oil, wine, bread and baked products. This possibility is reserved for individual farmers or co-operatives, registered in the company register according to Article 8 of the Law 29th December 1993 No. 580 (Italian Republic, 1993); who may sell directly inside and outside farm, products coming mainly from the respective farms, observing the current regulations regarding health and hygiene. All this should provide an instrument for rural and competitive development for the entire European agricultural production chain strongly influenced by the marketing conditions imposed by the mass retailing groups on their own suppliers. Not least is the possibility of creating work and occupation and adequately counteracting the phenomenon of the depopulation of the countryside, encouraging the return to agricultural activities on the part of young people; and, consequently, a form of safeguarding the environment by reducing the costs linked to hydro-geological instability and soil maintenance. This trend, together with the national directions, may represent a support even for small local farms which, taking advantage of simplified procedures consistent with the objectives of flexibility of the community Regulations (EC) (No. 852/2004; No. 853/2004) (European Commission, 2004; 2004a), may take part in the promotion of agricultural markets managed directly by the farmers as sales points for local products (farmers' markets), so as to guarantee a fairer price and consolidate the territorial link between production and consumption (short distribution chain or short circuit). Without, of course, renouncing the necessary prerequisites for placing any food on the market: health-hygiene; traceability; health and well-being of the animals; safeguarding of the environment and the plants.

Replication kinetics and cellular tropism of emerging reoviruses in sheep and swine respiratory ex vivo organ cultures.

Ex vivo organ cultures (EVOCs) are extensively used to study the cellular tropism and infectivity of different pathogens. In this study, we used ovine and porcine respiratory EVOCs to investigate the replication kinetics and cellular tropism of selected emerging reoviruses namely Pteropine orthoreovirus, an emerging bat-borne zoonotic respiratory virus, and atypical Bluetongue virus (BTV) serotypes which, unlike classical serotypes, do not cause Bluetongue, a major OIE-listed disease of ruminants. BTV failed to replicate in ovine EVOCs. Instead, PRV showed slight replication in porcine lower respiratory EVOCs and a more sustained replication in all ovine respiratory tissues. By confocal laser scanning microscopy, PRV was demonstrated to infect bronchiolar and type I pneumocytes of ovine tissues. Overall, respiratory EVOCs from different animal species, eventually obtained at slaughterhouse, are a useful tool for testing and preliminarily characterize novel and emerging viruses addressing the essential in vivo animal work. Further experiments are, indeed, warranted in order to characterize the pathogenesis and transmission of these emerging reoviruses.

Genome Sequence of Canine Adenovirus Type 1 Isolated from a Wolf (Canis lupus) in Southern Italy.

Canine adenovirus type 1 (CAdV-1), a DNA virus of the family Adenoviridae, causes infectious canine hepatitis, a highly contagious disease primarily affecting canids. In this report, we describe the isolation and whole-genome sequence of a CAdV-1 isolate from the liver of a free-ranging wolf (Canis lupus).

Coordinated Role of Toll-Like Receptor-3 and Retinoic Acid-Inducible Gene-I in the Innate Response of Bovine Endometrial Cells to Virus.

Bovine herpesvirus-4 (BoHV-4) and bovine viral diarrhea virus (BVDV) infect the uterus of cattle, often resulting in reduced fertility, or abortion of the fetus, respectively. Here, exposure of primary bovine endometrial cells to BoHV-4 or BVDV modulated the production of inflammatory mediators. Viral pathogen-associated molecular patterns (PAMPs) are detected via pattern-recognition receptors (PRRs). However, the relative contribution of specific PRRs to innate immunity, during viral infection of the uterus, is unclear. Endometrial epithelial and stromal cells constitutively express the PRR Toll-like receptor (TLR)-3, but, the status of retinoic acid-inducible gene I (RIG-I), a sensor of cytosolic nucleic acids, is unknown. Primary endometrial epithelial and stromal cells had low expression of RIG-I, which was increased in stromal cells after 12 h transfection with the TLR3 ligand Poly(I:C), a synthetic analog of double-stranded RNA. Furthermore, short interfering RNA targeting TLR3, or interferon (IFN) regulatory transcription factor 3, an inducer of type I IFN transcription, reduced Poly(I:C)-induced RIG-I protein expression and reduced inflammatory mediator secretion from stromal cells. We conclude that antiviral defense of endometrial stromal cells requires coordinated recognition of PAMPs, initially via TLR3 and later via inducible RIG-I.

Bovine herpesvirus 4-based vector delivering a hybrid rat/human HER-2 oncoantigen efficiently protects mice from autochthonous Her-2+ mammary cancer.

The epidermal growth factor receptor 2 (HER-2) oncogene is a major target for the immunotherapy of breast cancer. Following up to the therapeutic success achieved with Her-2-targeting monoclonal antibodies, immune-prophylactic approaches directed against Her-2 have also been investigated taking into account, and trying to overcome, Her-2 self-tolerance. Perhaps due to safety (and efficacy) concerns, the least explored anti-Her-2 active immunization strategy so far has been the one relying on viral-vectored vaccine formulations. Taking advantage of the favorable properties of bovine herpesvirus 4 (BoHV-4) in terms of safety and ease of manipulation as well as its previously documented ability to transduce and confer immunogenicity to heterologous antigens, we tested the ability of different recombinant HER-2-BoHV-4 immunogens to 8break tolerance and elicit a protective, anti-mammary tumor antibody response in HER-2 transgenic BALB-neuT mice. All the tested constructs expressed the HER-2 transgenes at high levels and elicited significant cellular immune responses in BALB/c mice upon administration via either DNA vaccination or viral infection. In BALB-neuT mice, instead, only the viral construct expressing the membrane-bound chimeric form of Her-2 protein (BoHV-4-RHuT-gD) elicited a humoral immune response that was more intense and earlier-appearing than that induced by DNA vaccination. In keeping with this observation, two administrations of BoHV-4-RHuT-gD effectively protected BALB-neuT mice from tumor formation, with 50% of vaccinated animals tumor-free after 30 weeks from immunization compared to 100% of animals exhibiting at least one palpable tumor in the case of animals vaccinated with the other BoHV-4-HER-2 constructs.

Aujeszky's disease in red fox (Vulpes vulpes): phylogenetic analysis unravels an unexpected epidemiologic link.

We describe Aujeszky's disease in a female of red fox (Vulpes vulpes). Although wild boar (Sus scrofa) would be the expected source of infection, phylogenetic analysis suggested a domestic rather than a wild source of virus, underscoring the importance of biosecurity measures in pig farms to prevent contact with wild animals.

Characterization of caprine herpesvirus 1 (CpHV1) glycoprotein E and glycoprotein I ectodomains expressed in mammalian cells.

Caprine herpesvirus 1 (CpHV1) is a member of ruminant alphaherpesviruses antigenically related to the prototype bovine herpesvirus 1 (BoHV1). Although cross reactivity between the two viruses involves many structural glycoproteins, the use of two competitive BoHV1 ELISAs detecting anti gB and gE antibodies has been proposed for CpHV1 infection, resulting mainly in a gB+/gE- reactivity and leading to suppose that CpHV1 gE may represent an useful target for the development of specific diagnostic test. Since CpHV1 gE gene has been only partially characterized so far, in this study the genome fragment of the short unique unit (Us) encompassing gI and gE gene was amplified and sequenced. Gene fragments encoding the ectodomain of both glycoproteins were subcloned into pSECTag2/Hygro and expressed in HEK293T cells as secreted form in serum free medium. Due to the lack of specific monoclonal antibodies (Mabs), the same recombinant glycoproteins were obtained from BoHV1 and used as positive control with a panel of specific gE and gI Mabs as well as in some ELISA assays. Results clearly indicate that the ectodomain of CpHV1 gE, immobilized on solid face in an indirect ELISA format, represents a sensitive and specific marker of infection, when compared with neutralization test, with absence of very low degree of cross-reactivity with BoHV1 gE counterpart, while the use of CpHV1 gI-ELISA or a combination of gE/gI complex did not significantly improve the sensitivity of the assay. In addition, in the rare event in which cross species barrier occurs for both viruses from their natural host to other species, the use of both BoHV1 and CpHV1 gE in a comparative assay may be proposed.