Spectral Profiling (Fourier Transform Infrared Spectroscopy) and Machine Learning for the Recognition of Milk from Different Bovine Breeds.

The Podolica cattle breed is widespread in southern Italy, and its productivity is characterized by low yields and an extraordinary quality of milk and meats. Most of the milk produced is transformed into "Caciocavallo Podolico" cheese, which is made with 100% Podolica milk. Fourier Transform Infrared Spectroscopy (FTIR) is the technique that, in this research work, was applied together with machine learning to discriminate 100% Podolica milk from contamination of other Calabrian cattle breeds. The analysis on the test set produced a misclassification percentage of 6.7%. Among the 15 non-Podolica samples in the test set, 2 were misclassified and recognized as Podolica milk even though the milk was from other species. The correct classification rate improved to 100% when the same method was applied to the recognition of Podolica and Pezzata Rossa milk produced by the same farm. Furthermore, this technique was tested for the recognition of Podolica milk mixed with milk from other bovine species. The multivariate model and the respective confusion matrices obtained showed that all the 14 Podolica samples (test set) mixed with 40% non-Podolica milk were correctly classified. In addition, Pezzata Rossa milk produced by the same farm was detected as a contaminant in Podolica milk from the same farm down to concentrations as little as 5% with a 100% correct classification rate in the test set. The method described yielded higher accuracy values when applied to the discrimination of milks from different breeds belonging to the same farm. One of the reasons for this phenomenon could be linked to the elimination of the environmental variable. However, the results obtained in this work demonstrate the possibility of using FTIR to discriminate between milks from different breeds.

The Bee Gut Microbiota: Bridging Infective Agents Potential in the One Health Context.

The bee gut microbiota plays an important role in the services the bees pay to the environment, humans and animals. Alongside, gut-associated microorganisms are vehiculated between apparently remote habitats, promoting microbial heterogeneity of the visited microcosms and the transfer of the microbial genetic elements. To date, no metaproteomics studies dealing with the functional bee microbiota are available. Here, we employ a metaproteomics approach to explore a fraction of the bacterial, fungal, and unicellular parasites inhabiting the bee gut. The bacterial community portrays a dynamic composition, accounting for specimens of human and animal concern. Their functional features highlight the vehiculation of virulence and antimicrobial resistance traits. The fungal and unicellular parasite fractions include environment- and animal-related specimens, whose metabolic activities support the spatial spreading of functional features. Host proteome depicts the major bee physiological activities, supporting the metaproteomics strategy for the simultaneous study of multiple microbial specimens and their host-crosstalks. Altogether, the present study provides a better definition of the structure and function of the bee gut microbiota, highlighting its impact in a variety of strategies aimed at improving/overcoming several current hot topic issues such as antimicrobial resistance, environmental pollution and the promotion of environmental health.

Mediterranean Plants with Antimicrobial Activity against Staphylococcus aureus, a Meta-Analysis for Green Veterinary Pharmacology Applications.

Antimicrobial resistance (AMR) has emerged as a global health crisis, necessitating the search for innovative strategies to combat infectious diseases. The unique biodiversity of Italian flora offers a treasure trove of plant species and their associated phytochemicals, which hold immense potential as a solution to address AMR. By investigating the antimicrobial properties of Italian flora and their phytochemical constituents, this study aims to shed light on the potential of phyto-complexes as a valuable resource for developing novel or supportive antimicrobial agents useful for animal production.

Recent Advances in the Determination of Milk Adulterants and Contaminants by Mid-Infrared Spectroscopy.

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 Functional Characteristics of Goat Cheese Microbiota from a One-Health Perspective.

Goat cheese is an important element of the Mediterranean diet, appreciated for its health-promoting features and unique taste. A pivotal role in the development of these characteristics is attributed to the microbiota and its continuous remodeling over space and time. Nevertheless, no thorough study of the cheese-associated microbiota using two metaomics approaches has previously been conducted. Here, we employed 16S rRNA gene sequencing and metaproteomics to explore the microbiota of a typical raw goat milk cheese at various ripening timepoints and depths of the cheese wheel. The 16S rRNA gene-sequencing and metaproteomics results described a stable microbiota ecology across the selected ripening timepoints, providing evidence for the microbiologically driven fermentation of goat milk products. The important features of the microbiota harbored on the surface and in the core of the cheese mass were highlighted in both compositional and functional terms. We observed the rind microbiota struggling to maintain the biosafety of the cheese through competition mechanisms and/or by preventing the colonization of the cheese by pathobionts of animal or environmental origin. The core microbiota was focused on other biochemical processes, supporting its role in the development of both the health benefits and the pleasant gustatory nuances of goat cheese.

Potential New Therapeutic Approaches Based on Punica granatum Fruits Compared to Synthetic Anthelmintics for the Sustainable Control of Gastrointestinal Nematodes in Sheep.

Parasites, in particular, gastrointestinal nematodes (GINs) represent one of the main burdens affecting small ruminant farming and pose a serious threat to their health, welfare, productivity, and reproduction. The correct management of animals and the correct use of anthelmintic drugs are the pillars of the GIN control programs for small ruminants. However, globally due to the indiscriminate use of synthetic anthelmintics, there is a significant increase in anthelmintic resistance phenomena to one or more classes of drugs. Even if such a problem never represented a serious threat in southern Italy because of the favourable environmental conditions and because of the good farm management, the phenomenon is actually showing a steep increasing trend and requires alternative treatment measures and constant monitoring. The use of phytotherapies is considered a valuable alternative approach for GIN control in small ruminants and could help with reducing the amount of synthetic drugs used and the forthcoming anthelmintic resistance. From this perspective, the Calabria territory offers a wide number of plants with anthelmintic efficacy that could be helpful for this purpose. The aim of this study was to evaluate the anthelmintic efficacy of aqueous pomegranate (Punica granatum L.) macerate compared to the treatment with Ivermectin and Albendazole in sheep naturally infected with GINs. The pomegranate macerate derives from the ethnoveterinary knowledge of the Calabria region, Southern Italy. The anthelmintic efficacy was evaluated according to the faecal egg count reduction test (FECRt) using the FLOTAC techniques in two sheep farms in Southern Italy. The FECR was calculated from individual samples using the formula FECR = 100 × (1 - [T2/C2]). The treatment with Albendazole in the first farm showed an efficacy of 99.8% after 14 days and 94.8% after 21 days, while the treatment with Ivermectin in the second farm showed an efficacy of 99.9% after 14 days and 96.5% after 21 days of treatment. The pomegranate macerate, in both farms, showed a value of efficacy of around 50% from day 7 to day 21 after the treatment. Previous studies highlighted the presence of gallic acid as the main component in the pomegranate macerate, and its efficacy in nematode control has been as well previously demonstrated in other plant extracts. This in vivo study demonstrated the unequivocal efficacy of plant macerate in easily reducing 50% of the number of GIN eggs in sheep faeces. These results, obtained without the use of synthetic anthelmintics, indicate the use of green veterinary pharmacology as a sustainable alternative to the use of synthetic drugs to reduce the increase in drug resistance phenomena and the environmental impact.

Plants with Antimicrobial Activity Growing in Italy: A Pathogen-Driven Systematic Review for Green Veterinary Pharmacology Applications.

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.

Environmental Impact of Pharmaceutical Pollutants: Synergistic Toxicity of Ivermectin and Cypermethrin.

Veterinary antiparasitic pharmaceuticals as well as pesticides have been detected in surface waters, and they may cause several toxic effects in this environmental compartment. In the present study, we evaluated the toxicity after exposure of different concentration of ivermectin (IVM; 50, 100, and 200 µg L-1) and cypermethrin (CYP; 5, 10, and 25 µg L-1) and the combination of these two compounds at non-toxic concentration (IVM 100 + CYP 5 µg L-1) in zebrafish embryos. Combination of IVM at 100 µg L-1 with CYP at 5 µg L-1 exposure induced hatching delay and malformations at 96 hpf in zebrafish larvae as well as significant induction of cell death in zebrafish larvae. At the same time, the two single concentrations of IVM and CYP did not show a toxic effect on zebrafish development. In conclusion, our study suggests that IVM and CYP show a synergistic effect at common, ineffective concentrations, promoting malformation and cell death in fish development.

Mid-infrared (MIR) spectroscopy for the detection of cow's milk in buffalo milk.

In Italy, buffalo mozzarella is a largely sold and consumed dairy product. The fraudulent adulteration of buffalo milk with cheaper and more available milk of other species is very frequent. In the present study, Fourier transform infrared spectroscopy (FTIR), in combination with multivariate analysis by partial least square (PLS) regression, was applied to quantitatively detect the adulteration of buffalo milk with cow milk by using a fully automatic equipment dedicated to the routine analysis of the milk composition. To enhance the heterogeneity, cow and buffalo bulk milk was collected for a period of over three years from different dairy farms. A total of 119 samples were used for the analysis to generate 17 different concentrations of buffalo-cow milk mixtures. This procedure was used to enhance variability and to properly randomize the trials. The obtained calibration model showed an R 2 ≥ 0.99 (R 2cal. = 0.99861; root mean square error of cross-validation [RMSEC] = 2.04; R 2val. = 0.99803; root mean square error of prediction [RMSEP] = 2.84; root mean square error of cross-validation [RMSECV] = 2.44) suggesting that this method could be successfully applied in the routine analysis of buffalo milk composition, providing rapid screening for possible adulteration with cow's milk at no additional cost.

Green Veterinary Pharmacology for Honey Bee Welfare and Health: Origanum heracleoticum L. (Lamiaceae) Essential Oil for the Control of the Apis mellifera Varroatosis.

Varroatosis, caused by the Varroa destructor mite, is currently the most dangerous parasitic disease threatening the survival of honey bees worldwide. Its adverse effect on the welfare and health of honey bees requires the regular use of specific acaricides. This condition has led to a growing development of resistance phenomena towards the most frequently used drugs. In addition, another important aspect that should not be understated, is the toxicity and persistence of chemicals in the environment. Therefore, the identification of viable and environmentally friendly alternatives is urgently needed. In this scenario, essential oils are promising candidates. The aim of this study was to assess the contact toxicity, the fumigation efficacy and the repellent effect of Origanum heracleoticum L. essential oil (EO) against V. destructor mite. In the contact tests, each experimental replicate consisted of 15 viable adult female mites divided as follows: 5 treated with EO diluted in HPLC grade acetone, 5 treated with acetone alone (as negative control) and 5 treated with Amitraz diluted in acetone (as positive control). The EO was tested at concentrations of 0.125, 0.25, 0.5, 1 and 2 mg/mL. For each experimental replicate, mortality was manually assessed after one hour. The efficacy of EO fumigation was evaluated through prolonged exposure at different time intervals. After each exposure, the 5 mites constituting an experimental replicate were transferred to a Petri dish containing a honey bee larva and mortality was assessed after 48 h. The repellent action was investigated by implementing a directional choice test in a mandatory route. During the repellency tests the behavior of the mite (90 min after its introduction in the mandatory route) was not influenced by the EO. In contact tests, EO showed the best efficacy at 2 and 1 mg/mL concentrations, neutralizing (dead + inactivated) 90.9% and 80% of the mites, respectively. In fumigation tests, the mean mortality rate of V. destructor at maximum exposure time (90 min) was 60% and 84% at 24 and 48 h, respectively. Overall, these results demonstrate a significant efficacy of O. heracleoticum EO against V. destructor, suggesting a possible alternative use in the control of varroatosis in honey bee farms in order to improve Apis mellifera welfare and health and, consequently, the hive productions.

In Vitro Anthelmintic Activity of Isatis tinctoria Extracts against Ewes' Gastrointestinal Nematodes (GINs), a Possible Application for Animal Welfare.

Sheep gastrointestinal nematode (GIN) infestation represents a limiting factor for sheep farming and milk production in Italy. The development of anthelmintic resistance to conventionally used drugs suggests the path towards the use of natural remedies as a possible alternative. The purpose of this study is to evaluate the in vitro anthelmintic efficacy of the hydroalcoholic extracts of basal leaves (It-BL), cauline leaves (It-CL) and flowers (It-F) of Isatis tinctoria (Brassicaceae), a spontaneous Sicilian species renowned as an important source of bioactive compounds. The dry extracts of the different parts of the plant were tested using the egg hatch test (EHT) in vitro to verify the efficacy against ovine GIN at different concentrations (1.00, 0.5, 0.25, 0.125 mg/mL). Thiabendazole and deionized water were used as positive and negative controls, respectively. The results obtained from EHT indicated that all the I. tinctoria extracts were highly effective (p < 0.0001) in inhibiting egg hatching within 48 h of exposure. The in vitro inhibitory effect was never less than 84% in all doses tested, and it was only slightly lower than the standard drug thiabendazole (95.6%). The current study documents the anthelmintic activity of I. tinctoria against sheep's GIN, suggesting its application as alternative natural method to limit the use of antiparasitic drugs.

LAP-MALDI MS coupled with machine learning: an ambient mass spectrometry approach for high-throughput diagnostics.

Large-scale population screening for early and accurate detection of disease is a key objective for future diagnostics. Ideally, diagnostic tests that achieve this goal are also cost-effective, fast and easily adaptable to new diseases with the potential of multiplexing. Mass spectrometry (MS), particularly MALDI MS profiling, has been explored for many years in disease diagnostics, most successfully in clinical microbiology but less in early detection of diseases. Here, we present liquid atmospheric pressure (LAP)-MALDI MS profiling as a rapid, large-scale and cost-effective platform for disease analysis. Using this new platform, two different types of tests exemplify its potential in early disease diagnosis and response to therapy. First, it is shown that LAP-MALDI MS profiling detects bovine mastitis two days before its clinical manifestation with a sensitivity of up to 70% and a specificity of up to 100%. This highly accurate, pre-symptomatic detection is demonstrated by using a large set of milk samples collected weekly over six months from approximately 500 dairy cows. Second, the potential of LAP-MALDI MS in antimicrobial resistance (AMR) detection is shown by employing the same mass spectrometric setup and similarly simple sample preparation as for the early detection of mastitis.

Entomopathogenic Fungi for Pests and Predators Control in Beekeeping.

The emergence of resistance to chemical drugs in beekeeping is becoming a phenomenon of widespread concern. One promising alternative to the use of chemicals is entomopathogenic organisms that are environmentally friendly and are capable of stopping the expression of resistance once it has evolved. In the recent past, the scientific community has carried out several experiments addressing the use of microbiological control agents. In particular, experimental studies using entomopathogenic fungi have had more success in honey bee research. With their adherence properties and their ability to digest the cuticle and overcome the host defense mechanism, they could be a suitable ingredient in bioacaricides. Several promising fungi have been identified in the search for effective means to control pest populations. The data obtained from the different experiments are interesting and often favorable to their use, but there are also conflicting results. The aim of this review is to describe the state of the art on the topic under investigation.

Ivermectin (IVM) Possible Side Activities and Implications in Antimicrobial Resistance and Animal Welfare: The Authors' Perspective.

Ivermectin has a wide number of many diverse functions. Certainly, it is irreplaceable for the treatment of parasitic pathologies in both human and veterinary medicine, and the latter represents the major field of its application. It has been called the "drug for the world's poor" because of its role as a saviour for those living on the margins of society, in underdeveloped areas afflicted by devastating and debilitating diseases, such as Onchocerciasis and Lymphatic filariasis. It showed huge, unexpected potential as an antibacterial (Chlamydia trachomatis and mycobacteria), and it has antiviral and anti-inflammatory properties. The research line described here is placed right in the middle of the investigation on the impact of this drug as an antimicrobial and an immunomodulator. Being a drug widely employed for mass administration, it is mandatory to broaden the knowledge of its possible interaction with bacterial growth and its generation of antimicrobial resistance. Equally, it is important to understand the impact of these drugs on the immune systems of animal species, e.g., horses and dogs, in which this drug is often used. More importantly, could immunomodulation and antibacterial activity promote both bacterial growth and the occurrence of resistance mechanisms?

Are Physicochemical Properties Shaping the Allergenic Potency of Plant Allergens?

This review searched for published evidence that could explain how different physicochemical properties impact on the allergenicity of food proteins and if their effects would follow specific patterns among distinct protein families. Owing to the amount and complexity of the collected information, this literature overview was divided in two articles, the current one dedicated to protein families of plant allergens and a second one focused on animal allergens. Our extensive analysis of the available literature revealed that physicochemical characteristics had consistent effects on protein allergenicity for allergens belonging to the same protein family. For example, protein aggregation contributes to increased allergenicity of 2S albumins, while for legumins and cereal prolamins, the same phenomenon leads to a reduction. Molecular stability, related to structural resistance to heat and proteolysis, was identified as the most common feature promoting plant protein allergenicity, although it fails to explain the potency of some unstable allergens (e.g. pollen-related food allergens). Furthermore, data on physicochemical characteristics translating into clinical effects are limited, mainly because most studies are focused on in vitro IgE binding. Clinical data assessing how these parameters affect the development and clinical manifestation of allergies is minimal, with only few reports evaluating the sensitising capacity of modified proteins (addressing different physicochemical properties) in murine allergy models. In vivo testing of modified pure proteins by SPT or DBPCFC is scarce. At this stage, a systematic approach to link the physicochemical properties with clinical plant allergenicity in real-life scenarios is still missing.

Are Physicochemical Properties Shaping the Allergenic Potency of Animal Allergens?

Key determinants for the development of an allergic response to an otherwise 'harmless' food protein involve different factors like the predisposition of the individual, the timing, the dose, the route of exposure, the intrinsic properties of the allergen, the food matrix (e.g. lipids) and the allergen modification by food processing. Various physicochemical parameters can have an impact on the allergenicity of animal proteins. Following our previous review on how physicochemical parameters shape plant protein allergenicity, the same analysis was proceeded here for animal allergens. We found that each parameter can have variable effects, ranging on an axis from allergenicity enhancement to resolution, depending on its nature and the allergen. While glycosylation and phosphorylation are common, both are not universal traits of animal allergens. High molecular structures can favour allergenicity, but structural loss and uncovering hidden epitopes can also have a similar impact. We discovered that there are important knowledge gaps in regard to physicochemical parameters shaping protein allergenicity both from animal and plant origin, mainly because the comparability of the data is poor. Future biomolecular studies of exhaustive, standardised design together with strong validation part in the clinical context, together with data integration model systems will be needed to unravel causal relationships between physicochemical properties and the basis of protein allergenicity.

Acid Hydrolysis of Gluten Enhances the Skin Sensitizing Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins.

SCOPE: Personal care products containing hydrolyzed gluten have been linked to spontaneous sensitization through the skin, however the impact of the hydrolysate characteristics on the sensitizing capacity is generally unknown. METHODS AND RESULTS: The physicochemical properties of five different wheat-derived gluten products (one unmodified, one enzyme hydrolyzed, and three acid hydrolyzed) are investigated, and the skin sensitizing capacity is determined in allergy-prone Brown Norway rats. Acid hydrolyzed gluten products exhibited the strongest intrinsic sensitizing capacity via the skin. All hydrolyzed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolyzed products is associated with product-specific sensitization in wheat tolerant rats. Sensitization to acid hydrolyzed gluten products is associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitization induced by unmodified gluten or enzyme hydrolyzed gluten. CONCLUSION: Acid hydrolysis enhances the skin sensitizing capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolyzed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals.

Green Veterinary Pharmacology Applied to Parasite Control: Evaluation of Punica granatum, Artemisia campestris, Salix caprea Aqueous Macerates against Gastrointestinal Nematodes of Sheep.

Resistance to anthelmintic drugs in gastrointestinal nematodes (GIN) of sheep is of high concern for livestock production worldwide. In Calabria (southern Italy), many plants have been used in ethnoveterinary medicine for parasite control in small ruminants. Here, we present an in vivo evaluation of anthelmintic efficacy of three plant extracts. The first was based on bark and leaves of Salix caprea, the second and the third were based on the whole plant Artemisia campestris and whole fruit (seeds and peel) of Punica granatum, respectively. Anthelmintic efficacy was evaluated according to the fecal egg count reduction test (FECRT) performed with the FLOTAC technique. The results showed a significant anthelmintic effect of Punica granatum macerate (50%), a low effectiveness of the Artemisia campestris macerate (20%), and a complete ineffectiveness of Salix caprea macerate (0.1%). With these outcomes, we report a P. granatum-based remedy reducing 50% GIN egg output. This result was obtained without using any synthetic drug, paving the way for the employment of green veterinary pharmacology (GVP) as a complementary and sustainable method to reduce the use of chemicals and to counteract anthelmintic resistance.

Parathyroid Carcinoma and Adenoma Co-existing in One Patient: Case Report and Comparative Proteomic Analysis.

BACKGROUND/AIM: The lack of specific parathyroid carcinoma (PC) biomarkers in clinical practice points out the importance of analyzing the proteomic signature of this cancer. We performed a comparative proteomic analysis of PC and parathyroid adenoma (PA) co-existing in the same patient. PATIENTS AND METHODS: PC and PA were taken from a 63-year-old patient. Using two-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry we examined the differences between PC and PA proteins. For validation, additional PC and PA samples were obtained from 10 patients. Western blot analysis was used to validate the difference of expression observed with 2D-DIGE analysis. Bioinfomatic analysis was performed using QIAGEN's Ingenuity Pathways Analysis (IPA) to determine the predominant canonical pathways and interaction networks involved. RESULTS: Thirty-three differentially expressed proteins were identified in PC compared to PA. Among these, ubiquitin C-terminal hydrolase-L1 (UCH-L1) was highly overexpressed in PC. The result was confirmed by Western Blot analysis in additional PC samples. CONCLUSION: Our comparative proteomic analysis of co-existing neoplasms allowed detecting specific and peculiar differences between PC and PA overcoming population biological variability.

Fungal Contamination and Aflatoxin B1 Detected in Hay for Dairy Cows in South Italy.

The growth of filamentous fungi on fodder is recognized as responsible for fungal deterioration and mycotoxin contamination of the plant mass leads to economic losses in the dairy cow production system. Mycotoxin contamination has significant implications for human and animal health and is one of the major concerns in the food and feed chain. This research provides an insight into the variety of viable molds (i.e., filamentous microfungi) that can be isolated from hay produced in South Italy and destined to dairy cows. On different lots of hay (n = 55) collected from 20 dairy farms, a total of 33 different fungal species were identified. The most representative was Cladosporium cladosporioides (n = 46, 84%) followed by Alternaria alternata (n = 25, 45%), and Rhizopus stolonifer (n = 24, 44%). The species most closely related to aflatoxin (AF) contamination, Aspergillus flavus, was often isolated (n = 11, 20%). Regarding AF detection, all the hay samples were found to be scarcely contaminated by AFB1 and showed values from 0.0020 to 0.0077 mg/kg, below the limits established by European Union (EU legislation) (0.02 mg/kg). None of the samples were positive for Aspergillia and tested for AFB1 showed results exceeding established limits. Additionally, hay with moisture between 15.0 and 19.2% or crude ash on dry matter content ranging from 14.0 to 15.5% reported an increased presence of AFB1 (p < 0.05) compared to the other samples. All the analyzed hay samples, besides the presence of molds, can be considered safe for the presence of AFB1. Prevention of mold spoilage is mandatory to reduce the exposure of humans and animals to mycotoxins.