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Abstract The role of gut microbiota in health and disease is being thoroughly examined in various contexts, with a specific focus on the bacterial fraction due to its significant abundance. However, despite their lower abundance, viruses within the gut microbiota are gaining recognition for their crucial role in shaping the structure and function of the intestinal microbiota, with significant effects on the host as a whole, particularly the immune system. Similarly, environmental factors such as stress are key in modulating the host immune system, which in turn influences the composition of the gut virome and neurological functions through the bidirectional communication of the gut–brain axis. In this context, alterations in the host immune system due to stress and/or dysbiosis of the gut virome are critical factors in the development of both infectious and noninfectious diseases. The molecular mechanisms and correlation patterns between microbial species are not yet fully understood. This literature review seeks to explore the interconnected relationship between stress and the gut virome, with a focus on how this interaction is influenced by the host's immune system. We also discuss how disturbances in this finely balanced system can lead to the onset and/or progression of diseases.

Zoonoses have rapidly spread globally, necessitating the implementation of vaccination strategies as a control measure. Emerging and re-emerging vector-borne diseases are among the major global public health concerns. Dengue, a zoonotic viral infection transmitted to humans by a vector, the Aedes mosquito, is a severe global health problem. Dengue is a serious tropical infectious disease, second only to malaria, causing around 25,000 deaths each year. The resurgence of Dengue is mainly due to climate change, demographic transitions and evolving social dynamics. The development of an effective vaccine against Dengue has proven to be a complex undertaking due to four different viral serotypes with distinct antigenic profiles. This review highlights the urgent need to address the dengue threat by exploring the application of biotechnological and -OMICS sciences.

The hepatitis E virus (HEV) has become increasingly important in recent years in terms of risk for public health, as the main causative agent of acute viral hepatitis. It is a foodborne disease transmitted to humans through the consumption of contaminated water or contaminated food. Human-to-human transmission is sporadic and is linked to transfusions or transplants. The main reservoirs of the hepatitis E virus are domestic pigs and wild boars, although, compared to pigs, wild boars represent a lesser source of risk since their population is smaller and the consumption of derived products is more limited. These peculiarities often make the role of the wild boar reservoir in the spread of the disease underestimated. As a public health problem that involves several animal species and humans, the management of the disease requires an interdisciplinary approach, and the concept of “One Health” must be addressed. In this direction, the present review intends to analyze viral hepatitis E, with a particular focus on wild boar. For this purpose, literature data have been collected from different scientific search engines: PubMed, MEDLINE, and Google scholar, and several keywords such as “HEV epidemiology”, “Extrahepatic manifestations of Hepatitis E”, and “HEV infection control measures”, among others, have been used. In the first part, the manuscript provides general information on the disease, such as epidemiology, transmission methods, clinical manifestations and implications on public health. In the second part, it addresses in more detail the role of wild boar as a reservoir and the implications related to the virus epidemiology. The document will be useful to all those who intend to analyze this infectious disease from a “One-Health” perspective.

Proteins involved in antibiotic resistance (resistome) and with antimicrobial activity are present in biological specimens. This study aims to explore the presence and abundance of antimicrobial peptides (AMPs) and resistome proteins in bovine milk from diverse breeds and from intensive (Pezzata rossa, Bruna alpina, and Frisona) and non-intensive farming (Podolica breeds). Liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) mass spectrometry (MS) profiling, bottom-up proteomics, and metaproteomics were used to comprehensively analyze milk samples from various bovine breeds in order to identify and characterize AMPs and to investigate resistome proteins. LAP-MALDI MS coupled with linear discriminant analysis (LDA) machine learning was employed as a rapid classification method for Podolica milk recognition against the milk of other bovine species. The results of the LAP-MALDI MS analysis of milk coupled with the linear discriminant analysis (LDA) demonstrate the potential of distinguishing between Podolica and control milk samples based on MS profiles. The classification accuracy achieved in the training set is 86% while it reaches 98.4% in the test set. Bottom-up proteomics revealed approximately 220 quantified bovine proteins (identified using the Bos taurus database), with cathelicidins and annexins exhibiting higher abundance levels in control cows (intensive farming breeds). On the other hand, the metaproteomics analysis highlighted the diversity within the milk’s microbial ecosystem with interesting results that may reflect the diverse environmental variables. The bottom-up proteomics data analysis using the Comprehensive Antibiotic Resistance Database (CARD) revealed beta-lactamases and tetracycline resistance proteins in both control and Podolica milk samples, with no relevant breed-specific differences observed.

Domestic chicken farming has been promoted and spread in several Italian municipalities and worldwide as an aid to the self-consumption of domestically produced food. This study investigated the levels of four toxic elements (As, Cd, Hg, and Pb) in eggs from an ethical laying hen farm, comparing the element concentrations with those possibly present in supermarket eggs. A total of 201 eggs, 141 from the farm and produced by different hen genotypes, and 60 from the supermarket, were collected. The levels of the toxic elements were evaluated in the yolk, albumen, and eggshells of all eggs. The results show that the supermarket eggs’ yolk and albumen were more contaminated with lead, compared to the rural eggs. Contrarily, the mean content of arsenic was higher in the albumen and eggshells of the rural eggs, compared to the supermarket eggs. The cadmium content was below the LOQ (0.005 mg/kg) in all samples. The mercury content was below or around the LOQ in all rural eggs. Overall, the supermarket egg albumens were significantly more contaminated than the rural ones. No significant differences were found in quality parameters for both types of eggs. The toxic element values that were detected were in line with other studies in the literature. However, despite the concentrations found not representing a risk to the consumers’ health, the results of this study raise a potential food safety issue, and it would be desirable to set specific MRLs for eggs for consumers’ protection.

ABSTRACT Patients with 2019 coronavirus disease (COVID-19) exhibit a broad spectrum of clinical presentations. A person’s antimicrobial antibody profile, as partially shaped by past infection or vaccination, can reflect the immune system health that is critical to control and resolve the infection. We performed an explorative immunoproteomics study using microbial protein arrays displaying 318 full-length antigens from 77 viruses and 3 bacteria. We compared antimicrobial antibody profiles between 135 patients with mild COVID-19 disease and 215 patients with severe disease in 3 independent cohorts from Mexico and Italy. Severe disease patients were older with higher prevalence of comorbidities. We confirmed that severe disease patients elicited a stronger anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) response. We showed that antibodies against HCoV-229E and HcoV-NL63 but not against HcoV-HKU1 and HcoV-OC43 were also higher in those who had severe disease. We revealed that for a set of IgG and IgA antibodies targeting coronaviruses, herpesviruses, and other respiratory viruses, a subgroup of patients with the highest reactivity levels had a greater incidence of severe disease compared to those with mild disease across all three cohorts. On the contrary, fewer antibodies showed consistent greater prevalence in mild disease in all 3 cohorts. IMPORTANCE The clinical presentations of COVID-19 range from asymptomatic to critical illness that may lead to intensive care or even death. The health of the immune system, as partially shaped by past infections or vaccinations, is critical to control and resolve the infection. Using an innovative protein array platform, we surveyed antibodies against hundreds of full-length microbial antigens from 80 different viruses and bacteria in COVID-19 patients from different geographic regions with mild or severe disease. We not only confirmed the association of severe COVID-19 disease with higher reactivity of antibody responses to SARS-CoV-2 but also uncovered known and novel associations with antibody responses against herpesviruses and other respiratory viruses. Our study represents a significant step forward in understanding the factors contributing to COVID-19 disease severity. We also demonstrate the power of comprehensive antimicrobial antibody profiling in deciphering risk factors for severe COVID-19. We anticipate that our approach will have broad applications in infectious diseases.

Antimicrobial resistance is an increasingly widespread phenomenon that is of particular concern because of the possible consequences in the years to come. The dynamics leading to the resistance of microbial strains are diverse, but certainly include the incorrect use of veterinary drugs both in terms of dosage and timing of administration. Moreover, the drug is often administered in the absence of a diagnosis. Many active ingredients in pharmaceutical formulations are, therefore, losing their efficacy. In this situation, it is imperative to seek alternative treatment solutions. Essential oils are mixtures of compounds with different pharmacological properties. They have been shown to possess the antibacterial, anti-parasitic, antiviral, and regulatory properties of numerous metabolic processes. The abundance of molecules they contain makes it difficult for treated microbial species to develop pharmacological resistance. Given their natural origin, they are environmentally friendly and show little or no toxicity to higher animals. There are several published studies on the use of essential oils as antimicrobials, but the present literature has not been adequately summarized in a manuscript. This review aims to shed light on the results achieved by the scientific community regarding the use of essential oils to treat the main agents of bacterial infection of veterinary interest in livestock. The Google Scholar, PubMed, SciELO, and SCOPUS databases were used for the search and selection of studies. The manuscript aims to lay the foundations for a new strategy of veterinary drug use that is more environmentally friendly and less prone to the emergence of drug resistance phenomena.

Antimicrobial resistance (AMR) is a complex and somewhat unpredictable phenomenon. Historically, the utilization of avoparcin in intensive farming during the latter part of the previous century led to the development of resistance to vancomycin, a crucial antibiotic in human medicine with life-saving properties. Currently, in the European Union, there is a growing reliance on the ionophore antibiotic monensin (MON), which acts both as a coccidiostat in poultry farming and as a preventative measure against ketosis in lactating cows. Although many researchers claim that MON does not induce cross-resistance to antibiotics of clinical relevance in human medicine, some conflicting reports exist. The numerous applications of MON in livestock farming and the consequent dissemination of the compound and its metabolites in the environment require further investigation to definitively ascertain whether MON represents a potential vector for the propagation of AMR. It is imperative to emphasize that antibiotics cannot substitute sound animal husbandry practices or tailored dietary regimens in line with the different production cycles of livestock. Consequently, a rigorous evaluation is indispensable to assess whether the economic benefits associated with MON usage justify its employment, also considering its local and global environmental ramifications and the potential risk of instigating AMR with increased costs for its control.

IntroductionBreast cancer (BC) is the leading cause of cancer-related death in women worldwide. Pathogenic variants in BRCA1 and BRCA2 genes account for approximately 50% of all hereditary BC, with 60-80% of patients characterized by Triple Negative Breast Cancer (TNBC) at an early stage phenotype. The identification of a pathogenic BRCA1/2 variant has important and expanding roles in risk-reducing surgeries, treatment planning, and familial surveillance. Otherwise, finding unclassified Variants of Unknown Significance (VUS) limits the clinical utility of the molecular test, leading to an “imprecise medicine”.MethodsWe reported the explanatory example of the BRCA1 c.5057A>C, p.(His1686Pro) VUS identified in a patient with TNBC. We integrated data from family history and clinic-pathological evaluations, genetic analyses, and bioinformatics in silico investigations to evaluate the VUS classification.ResultsOur evaluation posed evidences for the pathogenicity significance of the investigated VUS: 1) association of the BRCA1 variant to cancer-affected members of the family; 2) absence of another high-risk mutation; 3) multiple indirect evidences derived from gene and protein structural analysis.DiscussionIn line with the ongoing efforts to uncertain variants classification, we speculated about the relevance of an in-depth assessment of pathogenicity of BRCA1/2 VUS for a personalized management of patients with BC. We underlined that the efficient integration of clinical data with the widest number of supporting molecular evidences should be adopted for the proper management of patients, with the final aim of effectively guide the best prognostic and therapeutic paths.

The presence of chemical contaminants, toxins, or veterinary drugs in milk, as well as the adulteration of milk from different species, has driven the development of new tools to ensure safety and quality. Several analytical procedures have been proposed for the rapid screening of hazardous substances or the selective confirmation of the authenticity of milk. Mid-infrared spectroscopy and Fourier-transform infrared have been two of the most relevant technologies conventionally employed in the dairy industry. These fingerprint methodologies can be very powerful in determining the trait of raw material without knowing the identity of each constituent, and several aspects suggest their potential as a screening method to detect adulteration. This paper reviews the latest advances in applying mid-infrared spectroscopy for the detection and quantification of adulterants, milk dilution, the presence of pathogenic bacteria, veterinary drugs, and hazardous substances in milk.

The Varroa destructor parasite is the main obstacle to the survival of honey bee colonies. Pest control mainly involves the use of synthetic drugs which, used with the right criteria and in rotation, are able to ensure that infestation levels are kept below the damage threshold. Although these drugs are easy to use and quick to apply, they have numerous disadvantages. Their prolonged use has led to the emergence of pharmacological resistance in treated parasite populations; furthermore, the active ingredients and/or their metabolites accumulate in the beehive products with the possibility of risk for the end consumer. Moreover, the possibility of subacute and chronic toxicity phenomena for adult honeybees and their larval forms must be considered. In this scenario, eco-friendly products derived from plant species have aroused great interest over the years. In recent decades, several studies have been carried out on the acaricidal efficacy of plant essential oils (EOs). Despite the swarming of laboratory and field studies, however, few EO products have come onto the market. Laboratory studies have often yielded different results even for the same plant species. The reason for this discrepancy lies in the various study techniques employed as well as in the variability of the chemical compositions of plants. The purpose of this review is to take stock of the research on the use of EOs to control the V. destructor parasite. It begins with an extensive discussion of the characteristics, properties, and mechanisms of action of EOs, and then examines the laboratory and field tests carried out. Finally, an attempt is made to standardize the results and open up new lines of study in future.

Metabolic-dysfunction-associated fatty liver disease (MAFLD) is the recent nomenclature designation that associates the condition of non-alcoholic fatty liver disease (NAFLD) with metabolic dysfunction. Its diagnosis has been debated in the recent period and is generally associated with a diagnosis of steatosis and at least one pathologic condition among overweight/obesity, type 2 diabetes mellitus, and metabolic dysregulation. Its pathogenesis is defined by a “multiple-hit” model and is associated with alteration or dysbiosis of the gut microbiota. The pathogenic role of dysbiosis of the gut microbiota has been investigated in many diseases, including obesity, type 2 diabetes mellitus, and NAFLD. However, only a few works correlate it with MAFLD, although common pathogenetic links to these diseases are suspected. This review underlines the most recurrent changes in the gut microbiota of patients with MAFLD, while also evidencing possible pathogenetic links.

Abstract COVID-19 caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in Wuhan (Hubei province, China) during late 2019. It has spread across the globe affecting nearly 21 million people with a toll of 0.75 million deaths and restricting the movement of most of the world population during the past 6 months. COVID-19 became the leading health, economic, and humanitarian challenge of the twenty-first century. In addition to the considerable COVID-19 cases, hospitalizations, and deaths in humans, several cases of SARS-CoV-2 infections in animal hosts (dog, cat, tiger, lion, and mink) have been reported. Thus, the concern of pet owners is increasing. Moreover, the dynamics of the disease requires further explanation, mainly concerning the transmission of the virus from humans to animals and vice versa. Therefore, this study aimed to gather information about the reported cases of COVID-19 transmission in animals through a literary review of works published in scientific journals and perform genomic and phylogenetic analyses of SARS-CoV-2 isolated from animal hosts. Although many instances of transmission of the SARS-CoV-2 have been reported, caution and further studies are necessary to avoid the occurrence of maltreatment in animals, and to achieve a better understanding of the dynamics of the disease in the environment, humans, and animals. Future research in the animal–human interface can help formulate and implement preventive measures to combat the further transmission of COVID-19.

Brucellosis caused by Brucella melitensis is a zoonosis frequently reported in the Mediterranean and Middle-East regions and responsible for important economic losses and reduced animal welfare. To date, current strategies applied to control or eradicate the disease relies on diagnostic tests that suffer from limited specificity in non-vaccinated animals; while prophylactic measures, when applied, use a live attenuated bacterial strain characterized by residual virulence on adult pregnant animals and difficulties in distinguishing vaccinated from infected animals. To overcome these issues, studies are desired to elucidate the bacterial biology and the pathogenetic mechanisms of both the vaccinal strain and the pathogenic strains. Proteomics has a potential in tackling issues of One-Health concern; here, we employed label-free shotgun proteomics to investigate the protein repertoire of the vaccinal strain B. melitensis Rev.1 and compare it with the proteome of the Brucella melitensis 16 M, a reference strain representative of B. melitensis field strains. Comparative proteomics profiling underlines common and diverging traits between the two strains. Common features suggest the potential biochemical routes responsible for the residual virulence of the vaccinal strain, whilst the diverging traits are suggestive biochemical signatures to be further investigated to provide an optimized diagnostic capable of discriminating the vaccinated from infected animals. The data presented in this study are openly available in PRIDE data repository at https://www.ebi.ac.uk/pride/, reference number PXD022472.

Among biogenic amines, histamine is most frequently involved in foodborne intoxication. To evaluate histamine formation in tuna, several storage conditions were reproduced. An LC-MS/MS method was used for analytical determinations. Fresh tuna samples (not contaminated and grafted with tuna muscle naturally incurred with histamine at 6000 mg/kg) were stored at 4, 12, and 20 °C, and daily samples were collected for 6 days. The development of histamine was observed only in grafted tuna samples. At 4 °C, histamine formation progressed from 12.8 mg/kg (day 1) up to 68.2 mg/kg (day 6). At 12 °C, higher concentrations developed (23.9 mg/kg on day 1 up to 2721.3 mg/kg on day 6) relative to 20 °C (from 12.0 to 1681.0 mg/kg). It was found that at 4 °C, if grafted tuna was submerged in oil, histamine formation progressed more slowly. In a naturally contaminated sample, it was observed that the histamine distribution was uniform, while the normal cooking process did not affect the histamine level. Furthermore, it was found that the use of histamine-contaminated equipment for food handling may result in histamine formation in food. These results confirm the importance of implementing good hygiene practices and respecting the cold chain.

Varroatosis is an important parasitic disease of Apis mellifera caused by the mite Varroa destructor (V. destructor). The parasite is able to transmit numerous pathogens to honeybees which can lead to colony collapse. In recent years, the effectiveness of authorized drug products has decreased due to increasing resistance phenomena. Therefore, the search for alternatives to commercially available drugs is mandatory. In this context, essential oils (EOs) prove to be a promising choice to be studied for their known acaricide properties. In this research work, the acaricide activity of EO vapours isolated from the epigeal part (whole plant) of fennel (Foeniculum vulgare sbps. piperitum) and its three fractions (leaves, achenes and flowers) against V. destructor was evaluated. The effectiveness of fumigation was studied using two methods. The first involved prolonged exposure of mites to oil vapour for variable times. After exposure, the five mites in each replicate were placed in a Petri dish with an Apis mellifera larva. Mortality, due to chronic toxicity phenomena, was assessed after 48 h. The second method aimed to translate the results obtained from the in vitro test into a semi-field experiment. Therefore, two-level cages were set up. In the lower compartment of the cage, a material releasing oil vapours was placed; in the upper compartment, Varroa-infested honeybees were set. The results of the first method showed that the increase in mortality was directly proportional to exposure time and concentration. The whole plant returned 68% mortality at the highest concentration (2 mg/mL) and highest exposure time (48 h control), while the leaves, achenes and flowers returned 64%, 52% and 56% mortality, respectively. In the semi-field experiment, a concentration up to 20 times higher than the one used in the in vitro study was required for the whole plant to achieve a similar mite drop of >50%. The results of the study show that in vitro tests should only be used for preliminary screening of EO activity. In vitro tests should be followed by semi-field tests, which are essential to identify the threshold of toxicity to bees and the effective dose to be used in field studies.

Animal productions, and populations, have been rapidly expanding over the last decades, forcing the ever-closer coexistence of human beings and domestic animals on our yet “narrow” planet [...]

The poultry red mite Dermanyssus gallinae is the most important blood-sucking ectoparasite in egg-laying hen facilities. The aim of this study was to evaluate the in vitro acaricidal activity of different concentration of authorized (phoxim, ByeMite®, 500 mg/mL) and unauthorized (fipronil, Frontline® 250 mg/100 mL spray) molecules on 14 field isolates of D. gallinae collected from different ornamental poultry farms from different Italian regions. The sensitivity test was performed by contact exposure to four concentrations of each insecticide diluted at 1:5 (10,000-2000-400-80 ppm for phoxim, 500-100-20-4 ppm for fipronil) on a filter paper. The effectiveness of the treatment was significantly (p < 0.0001) associated with the dose of the pesticide used. Considering the mean lethality, phoxim has greater efficacy compared to fipronil (p < 0.001). A great variability of lethality rate was observed with the increase in fipronil dilution; conversely, for phoxim, some outliers were observed, particularly in one farm, suggesting the hypothesis that a certain degree of resistance in the mite population could occur possibly as a consequence of the continual contact with the molecule. This underlines the importance of the use of licensed products administered at correct dosages and the need for alternative molecules to avoid the onset of drug resistance phenomena.

Drug resistance threatening humans may be linked with antimicrobial and anthelmintic resistance in other species, especially among farm animals and, more in general, in the entire environment. From this perspective, Green Veterinary Pharmacology was proven successful for the control of parasites in small ruminants and for the control of other pests such as varroa in bee farming. As in anthelmintic resistance, antimicrobial resistance (AMR) represents one of the major challenges against the successful treatment of infectious diseases, and antimicrobials use in agriculture contributes to the spread of more AMR bacterial phenotypes, genes, and proteins. With this systematic review, we list Italian plants with documented antimicrobial activity against possible pathogenic microbes. Methods: The literature search included all the manuscripts published since 1990 in PubMed, Web of Science, and Scopus using the keywords (i) “antimicrobial, plants, Italy”; (ii) “antibacterial, plant, Italy”; (iii) “essential oil, antibacterial, Italy”; (iv) “essential oil, antimicrobial, Italy”; (v) “methanol extract, antibacterial, Italy”; (vi) “methanol extract, antimicrobial, Italy”. Results: In total, 105 manuscripts that documented the inhibitory effect of plants growing in Italy against bacteria were included. One hundred thirty-five plants were recorded as effective against Gram+ bacteria, and 88 against Gram−. This will provide a ready-to-use comprehensive tool to be further tested against the indicated list of pathogens and will suggest new alternative strategies against bacterial pathogens to be employed in Green Veterinary Pharmacology applications.

Varroa destructor is the most dangerous pest that poses a threat to honey bee survival. In recent years, increasingly worrying phenomena of drug resistance have occurred to various active ingredients of pharmaceutical formulations used to control this parasitosis. Determining the level of infestation is essential to preventing the inappropriate use and abuse of veterinary medicines, and to choose the most appropriate time for treatment. This comparative study investigates the sensitivity and diagnostic accuracy of two field techniques for diagnosing V. destructor infestations in hives. The EasyCheck device (Véto-pharma) was used in two of its application modes, namely, the sugar roll test and carbon dioxide (CO2) injection. The experiments were conducted on 15 samples of 300 bees each taken from the same frame and checked for the presence of mites using standard and modified field techniques in both uncaged and caged queen hive conditions. The results demonstrate that the sugar roll technique is significantly more effective and safer than CO2 injection, allowing for a higher accuracy in diagnosing a V. destructor infestation. Furthermore, the evaluation of mites present on bees in brood block conditions has proven to be particularly reliable. Considering the number of mites on the filter of the device as an additional step helps to implement the diagnostic accuracy of the CO2 injection technique, however, not achieving the efficacy results of the sugar roll.

In fish-based foods, one of the effects of inappropriate storage can be the formation of biogenic amines. Among these, histamine is considered one of the most toxic. The purpose of the present study is to assess the occurrence of histamine in fish-based pet foods, and to evaluate the changes in histamine content during storage at different temperatures. For the analysis, an LC-MS/MS method was used. Fifty-eight pet foods were purchased, and an aliquot of them was analyzed just after opening the packages. Histamine was detected in 12 samples and concentrations ranged from 1.5 to 30.1 mg/kg. The remaining of each positive sample was divided into seven subsamples. One of them was used as a control sample and kept at −20 °C, while the other six were exposed to different environmental conditions. Samples exposed to room temperature showed no significant changes in histamine levels, while samples exposed to high temperatures showed significant increases in histamine content. Finally, samples exposed to refrigerator temperature showed a slight decrease in histamine levels. Under the experimental conditions, the EU limit of 100 mg/kg established for fishery products was never exceeded. These results seem to indicate a low risk of histamine intoxication in cats fed fish-based pet food.

Medicinal plants may be effective against helminthic infestation in animals, but to date few studies have investigated the real impact of anthelminthic medicinal plants in veterinary ethnopharmacology.The aim of this study was to assess the geographical use of anthelminthic medicinal plants in livestock in European Union (EU), and to quantify the anthelminthic efficacy of medicinal plants in comparison with anthelminthic drugs. Surveys on the use of anthelminthic traditional medicinal plants in livestock in the EU were included in the qualitative synthesis. Studies that investigated the efficacy of anthelminthic traditional medicinal plants in animals, compared with negative control and/or anthelminthic drugs, were included in the quantitative synthesis (network meta-analysis).Twelve surveys (9 in Italy, 2 in Spain, 1 in Austria) reported the use of anthelminthic medicinal plants in livestock living in EU Countries. Data obtained from 256 animals and extracted from 6 studies were included in the network meta-analysis. Medicinal plants and drugs were more effective than negative control (standardized mean difference [SMD]: -0.60 95%CrI -0.88 to -0.31, -0.73 95%CrI -1.08 to -0.38, respectively, P < 0.001). Overall, no difference was detected between anthelminthic medicinal plants and anthelminthic drugs, namely albendazole, ivermectin, fenbendazole, and doramectin (SMD: 0.26 95%CrI -0.02 to 0.55, P > 0.05). The most effective anthelminthic medicinal plants were Artemisia absintihium, Allium sativum, and Duranta erecta.There is the strong medical need of performing adequately powered randomized controlled trials in different livestock species aimed to improve the quality of the current evidence concerning the anthelminthic efficacy of medicinal plants compared to that of the currently available antiparasitic drugs.

Antibiotic resistance, and, in a broader perspective, antimicrobial resistance (AMR), continues to evolve and spread beyond all boundaries. As a result, infectious diseases have become more challenging or even impossible to treat, leading to an increase in morbidity and mortality. Despite the failure of conventional, traditional antimicrobial therapy, in the past two decades, no novel class of antibiotics has been introduced. Consequently, several novel alternative strategies to combat these (multi-) drug-resistant infectious microorganisms have been identified. The purpose of this review is to gather and consider the strategies that are being applied or proposed as potential alternatives to traditional antibiotics. These strategies include combination therapy, techniques that target the enzymes or proteins responsible for antimicrobial resistance, resistant bacteria, drug delivery systems, physicochemical methods, and unconventional techniques, including the CRISPR-Cas system. These alternative strategies may have the potential to change the treatment of multi-drug-resistant pathogens in human clinical settings.

The detection of Ochratoxin A (OTA) in the milk of ruminants occurs infrequently and at low levels, but its occurrence may be higher in dairy products such as cheese. The aim of this study was to investigate the presence of OTA in cheeses purchased in the metropolitan city of Bologna (Italy) and the surrounding area. For the analysis, a LC-MS/MS method with a limit of quantification (LOQ) of 1 µg/kg was used. OTA was detected in seven out of 51 samples of grated hard cheese (concentration range 1.3–22.4 µg/kg), while it was not found in the 33 cheeses of other types which were also analysed. These data show a low risk of OTA contamination for almost all types of cheese analysed. To improve the safety of cheese marketed in grated form, more regulations on cheese rind, which is the part most susceptible to OTA-producing moulds, should be implemented or, alternatively, producers should consider not using the rind as row material for grated cheese. It would be interesting to continue these investigations particularly on grated hard cheeses to have more data to update the risk assessment of OTA in cheese, as also suggested by EFSA in its 2020 scientific opinion on OTA.

A 6-year-old female goose (Branta hutchinsii) from a group of ornamental exotic geese was found dead due to severe respiratory failure, followed by emission of haemorrhagic sputum and blood clots from the beak and nostrils, and then collapse. At necropsy, the cause of death was attributed to a total of 76 helminth parasites found in the trachea and lungs, then identified as Cyathostoma bronchialis. The flock was initially treated by feed with flubendazole (1200 g/1000 kg/feed) for 7 consecutive days but, at the reappearance of the respiratory symptoms 10 days after, the animals were given fenbendazole by drinking water (300 mg/L) for 7 consecutive days, but at the reappearance of the respiratory symptoms 10 days after, the animals were given fenbendazole via drinking water (300 mg/L) for 7 consecutive days. Despite these treatments, the respiratory symptoms continued to relapse 10–15 days after the end of drug administration. In the literature, there are no data regarding drugs for the treatment of C. bronchialis infestations in geese, and the use of these drugs in exotic birds occurs as “off-label” use. This case study provides information on C. bronchialis life cycle, which is still poorly studied and poorly documented today. In particular, the case provides useful suggestions for evaluating an appropriate protocol for the treatment of C. bronchialis in geese.

Prosthetic joint replacement failure has a huge impact on quality of life and hospitalization costs. A leading cause of prosthetic joint infection is bacteria-forming biofilm on the surface of orthopedic devices. Staphylococcus epidermidis is an emergent, low-virulence pathogen implicated in chronic infections, barely indistinguishable from aseptic loosening when embedded in a mature matrix. The literature on the behavior of quiescent S. epidermidis in mature biofilms is scarce. To fill this gap, we performed comparative analysis of the whole proteomic profiles of two methicillin-resistant S. epidermidis strains growing in planktonic and in sessile form to investigate the molecular mechanisms underlying biofilm stability. After 72-h culture of biofilm-forming S. epidermidis, overexpression of proteins involved in the synthesis of nucleoside triphosphate and polysaccharides was observed, whereas planktonic bacteria expressed proteins linked to stress and anaerobic growth. Cytological analysis was performed to determine why planktonic bacteria unexpectedly expressed proteins typical of sessile culture. Images evidenced that prolonged culture under vigorous agitation can create a stressful growing environment that triggers microorganism aggregation in a biofilm-like matrix as a mechanism to survive harsh conditions. The choice of a unique late time point provided an important clue for future investigations into the biofilm-like behavior of planktonic cells. Our preliminary results may inform comparative proteomic strategies in the study of mature bacterial biofilm. Finally, there is an increasing number of studies on the aggregation of free-floating bacteria embedded in an extracellular matrix, prompting the need to gain further insight into this mode of bacterial growth.

Antimicrobial resistance (AMR) represents one of the most critical challenges that humanity will face in the following years. In this context, a “One Health” approach with an integrated multidisciplinary effort involving humans, animals and their surrounding environment is needed to tackle the spread of AMR. One of the most common ways for bacteria to live is to adhere to surfaces and form biofilms. Staphylococcus aureus (S. aureus) can form biofilm on most surfaces and in a wide heterogeneity of environmental conditions. The biofilm guarantees the survival of the S. aureus in harsh environmental conditions and represents an issue for the food industry and animal production. The identification and characterization of biofilm-related proteins may provide interesting insights into biofilm formation mechanisms in S. aureus. In this regard, the aims of this study were: (i) to use proteomics to compare proteomes of S. aureus growing in planktonic and biofilm forms in order to investigate the common features of biofilm formation properties of different strains; (ii) to identify specific biofilm mechanisms that may be involved in AMR. The proteomic analysis showed 14 differentially expressed proteins among biofilm and planktonic forms of S. aureus. Moreover, three proteins, such as alcohol dehydrogenase, ATP-dependent 6-phosphofructokinase, and fructose-bisphosphate aldolase, were only differentially expressed in strains classified as high biofilm producers. Differentially regulated catabolites metabolisms and the switch to lower oxygen-related metabolisms were related to the sessile conformation analyzed.

The environment, including animals and animal products, is colonized by bacterial species that are typical and specific of every different ecological niche. Natural and human-related ecological pressure promotes the selection and expression of genes related to antimicrobial resistance (AMR). These genes might be present in a bacterial consortium but might not necessarily be expressed. Their expression could be induced by the presence of antimicrobial compounds that could originate from a given ecological niche or from human activity. In this work, we applied (meta)proteomics analysis of bacterial compartment of raw milk in order to obtain a method that provides a measurement of circulating AMR involved proteins and gathers information about the whole bacterial composition. Results from milk analysis revealed the presence of 29 proteins/proteoforms linked to AMR. The detection of mainly β-lactamases suggests the possibility of using the milk microbiome as a bioindicator for the investigation of AMR. Moreover, it was possible to achieve a culture-free qualitative and functional analysis of raw milk bacterial consortia.

Aflatoxin M1 (AFM1) is a well-known mycotoxin that can be found in the milk of animals that have ingested feed contaminated with aflatoxin B1 (AFB1). In Italy, the development of donkey farms is mainly due to growing request of donkey milk, which is considered an incomparable substitute for human mother’s milk for its chemical composition and organoleptic characteristics. The aim of this study was to assess the occurrence of AFM1 in donkey milk produced in a farm in Northern Italy, also in view of the few data available about the presence of this mycotoxin in this type of milk. Therefore, 63 milk samples were collected and analyzed using a fast and sensitive HPLC and fluorescence detection (FLD) method previously optimized and validated. None of the milk samples collected were found to be contaminated at a level above the limit of quantification (LOQ) (0.0125 ng/mL), while only one sample showed traces of the mycotoxin at a concentration between the limit of detection (LOD) and LOQ (0.0044 ng/mL), well below the legal limit established for infant milk and follow-on milk (0.025 ng/mL). These results are in line with those of the few similar surveys carried out on donkey milk and seem to indicate a low risk of AFM1 contamination for this food.

Paratuberculosis is an infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP is an intracellular pathogen with a possible zoonotic potential since it has been successfully isolated from the intestine and blood of Crohn’s disease patients.Since no cure is available, after the detection of the disease, animal culling is the sole applicable containment strategy. However, the difficult detection of the disease in its subclinical form, facilitates its spread raising the need for the development of effective diagnosis and vaccination strategies. The prompt identification and isolation of the infected animals in the subclinical stage would prevent the spread of the infection.In the present study, an immunoinformatic approach has been used to investigate the immunogenic properties of 10 MAP proteins. These proteins were chosen according to a previously published immunoproteomics approach. For each previously-described immunoreactive protein, we predicted the epitopes capable of eliciting an immune response by binding both B-cells and/or class I MHC antigens. The retrieved peptide sequences were analyzed for their specificity and cross-reactivity. The final aim is to employ the discovered peptides sequences as a filtered library useful for early-stage diagnosis and/or to be used in novel multi-subunit or recombinant vaccine formulations.

Rapid profiling of the biomolecular components of milk can be useful for food quality assessment and for food fraud detection. Differences in commercial value and availability of milk from specific species are often the reasons for the illicit and fraudulent sale of milk whose species origin is wrongly declared. In this study, a fast, MS-based speciation method is presented to distinguish sheep from goat milk and sheep colostrum at different phases. Using liquid atmospheric pressure (AP)-matrix-assisted laser desorption/ionisation (MALDI) MS, it was possible to classify samples of goat and sheep milk with 100% accuracy in one minute of data acquisition per sample. Moreover, an accuracy of 98% was achieved in classifying pure sheep milk samples and sheep milk samples containing 10% goat milk. Evaluating colostrum quality and postnatal stages represents another possible application of this technology. Classification of sheep colostrum samples that were collected within 6 hours after parturition and 48 hours later was achieved with an accuracy of 84.4%. Our data show that substantial changes in the lipid profile can account for the accurate classification of colostrum collected at the early and late time points. This method applied to the analysis of protein orthologs of different species can, as in this case, allow unequivocal speciation analysis.

In this study comparative proteomics was used to define changes in the expression of the spermatozoa proteins during liquid storage. Semen from eight boars was analyzed on the day of collection and after liquid preservation at 15−17 °C for three days. Sperm parameters (concentration, motility, morphology, vitality) and percentage of non-capacitated and acrosomal-reacted spermatozoa were determined. Sperm proteins were extracted and separated by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteomic profiles were computationally compared to highlight differentially expressed protein spots that were, in turn, identified by mass spectrometry. The intensities of four spots were significantly different between fresh and liquid stored sperm. Namely: ATP citrate lyase, chaperonin containing T-complex polypeptide 1 (TCP1) subunit ε and probable phospholipid-transporting ATP-ase were over-expressed in liquid stored sperm, whereas cytosolic non-specific dipeptidase was over-expressed in fresh sperm. These differentially expressed proteins could be used as plausible biomarkers for the evaluation of boar semen quality and spermatozoa survival after liquid storage and could help to address problems associated with sperm preservation.

Ochratoxin A (OTA) is a mycotoxin that may be present in various food and feed of plant and animal origin, including chicken meat. In Italy, backyard poultry farming is rather widespread. Animals are raised for meat, eggs and for ornamental purpose, and they are often fed with home-made diets not subject to official controls. The purpose of this study was to evaluate exposure of ornamental and backyard chickens to OTA using biliary ochratoxin A as a biomarker. Therefore, bile samples, in addition to kidney, liver and muscle, were collected from 102 chickens reared in 16 farms located in 6 Italian regions. High-performance liquid chromatography method and fluorimetric detection (HPLC-FLD) analysis were carried out firstly on bile from all animals, and OTA was detected in 12 chickens (concentration range 3.83−170.42 µg/L). Subsequently, the kidneys of these chickens were also analysed, and the mycotoxin was not detected. The analytical detection limits (LODs) of OTA in bile and kidney were 2.1 µg/L and 0.1 µg/kg, respectively. In conclusion, these animals were exposed to OTA but their meat can be considered safe, given that this mycotoxin, if present, concentrates highest in kidneys. Biliary ochratoxin A confirms its use as a valid biomarker to assess exposure of poultry to OTA.

Fat mobilization in high-yielding dairy cows during early lactation occurs to overcome negative energy balance (NEB), caused by insufficient feed intake and the concomitant increased nutritional requirements. For this reason, adipose tissue represents an essential organ for healthy and performant lactation. However, only a few data are known about adipose tissue proteome and its metabolic status during peripartum. The aim of this study was to analyze the differential proteomics profiles of subcutaneous adipose tissue belonging to cows with different NEB scores (low NEB and severe NEB). Both groups were analyzed at three different time points (one month before calving, one and sixteen weeks after calving) that were related to different levels and rates of adipose tissue mobilization. The dataset highlighted the differential expression of the same four key proteins (annexin A2, actin-related protein 10, glyceraldehyde-3-phosphate dehydrogenase, and fatty acid-binding protein) involved in lipid metabolism during all time points and of other 22 proteins typical of the other comparisons among remaining time points. The obtained dataset suggested that the individual variability in adipose tissue metabolism/mobilization/energy availability could be linked to the different outcomes in levels of energy balance and related physical complications among dairy cows during peripartum.