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eDNA metabarcoding has been increasingly employed in the monitoring of marine invertebrate non-indigenous species (NIS), in particular using filtered seawater. However, comprehensive detection of all NIS may require a diversity of sampling substrates. To assess the effectiveness of 5 sample types (hard and artificial substrates, water, zooplankton) on the recovery of invertebrates' diversity, two marinas were monitored over three time points, using COI and 18S rRNA genes as DNA metabarcoding markers. We detected a total of 628 species and 23 NIS, with only up to 9% species and 17% of NIS detected by all sample types. Hard and artificial substrates were similar to each other but displayed the most significant difference in invertebrate recovery when compared to water eDNA and zooplankton. Five NIS are potential first records for Portugal. No NIS were detected in all sample types and seasons, highlighting the need for varied sampling approaches, and consideration of temporal variation for comprehensive marine NIS surveillance.
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Biodiversidade , Código de Barras de DNA Taxonômico , Invertebrados , Animais , Invertebrados/genética , Espécies Introduzidas , Portugal , RNA Ribossômico 18S/genética , Monitoramento Ambiental/métodos , Organismos Aquáticos/genéticaRESUMO
The therapeutic efficacy of camptothecin (CPT), a potent antitumor alkaloid, is hindered by its hydrophobic nature and instability, limiting its clinical use in treating cutaneous squamous cell carcinoma (SCC). This study introduces a novel nano drug delivery system (NDDS) utilizing functionalized mesoporous silica nanoparticles (FMSNs) for efficient CPT delivery. The FMSNs were loaded with CPT and subsequently coated with chitosan (CS) for enhanced stability and bioadhesion. Importantly, CpG oligodeoxynucleotide (CpG ODN) was attached onto the CS-coated FMSNs to leverage the immunostimulatory properties of CpG ODN, augmenting the chemotherapy's efficacy. The final formulation FMSN-CPT-CS-CpG displayed an average size of 241 nm and PDI of 0.316 with an encapsulation efficiency of 95 %. Comprehensive in vitro and in vivo analyses, including B16F10 cells and DMBA/TPA-induced SCC murine model, demonstrated that the FMSN-CPT-CS-CpG formulation significantly enhanced cytotoxicity against B16F10 cells and induced complete regression in 40 % of the in vivo subjects, surpassing the efficacy of standard CPT and FMSN-CPT treatments. This study highlights the potential of combining chemotherapeutic and immunotherapeutic agents in an NDDS for targeted, efficient skin cancer treatment.
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Camptotecina , Quitosana , Nanopartículas , Oligodesoxirribonucleotídeos , Dióxido de Silício , Neoplasias Cutâneas , Animais , Dióxido de Silício/química , Dióxido de Silício/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/química , Neoplasias Cutâneas/tratamento farmacológico , Nanopartículas/química , Camptotecina/administração & dosagem , Camptotecina/química , Camptotecina/farmacologia , Linhagem Celular Tumoral , Camundongos , Quitosana/química , Quitosana/administração & dosagem , Feminino , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Porosidade , Camundongos Endogâmicos C57BL , Portadores de Fármacos/química , Carcinoma de Células Escamosas/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacosRESUMO
This work aimed to investigate the impact of hydrogen peroxide (HP) punctual dosage on paracetamol (PCT) degradation, through Fenton and photo-Fenton processes under near-neutral pH conditions, using ferrioxalate and artificial sunlight. The assays were performed using a D-optimal experimental design, to statistically evaluate the influence of radiation (ON or OFF), HP concentration (94.5-756 mg L-1), and HP dosage (YES or NO), on PCT conversion. The optimal conditions determined from the study were: HP = 378 mg L-1, DOS = YES, and RAD = ON, achieving a predicted PCT conversion of 99.68% in 180 min. This result obtained from the model was very close to the experimental one (98.80%). It was verified that HP dosage positively influenced the iron catalytic cycle since higher Fe2+ concentrations were reached at shorter reaction times, accelerating not only PCT conversion but also its by-products hydroquinone and 1,4-benzoquinone, leading to better performances of Fenton and photo-Fenton reactions. Under optimal conditions and employing real water matrices (an artificial matrix with inorganic anions, a real groundwater sample, and a synthetic industrial wastewater), HP dosage demonstrated the ability to mitigate the negative effects caused by the content of different ions and other organic compounds and significantly improve HP consumption in challenging wastewater conditions.
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Acetaminofen , Oxalatos , Poluentes Químicos da Água , Acetaminofen/química , Peróxido de Hidrogênio/química , Águas Residuárias , Poluentes Químicos da Água/análise , OxirreduçãoRESUMO
Zooplankton are key components of estuarine trophic networks. However, routine monitoring is hindered by the difficulty of morphology-based identification. DNA-based methods allow us to circumvent some of these hurdles, providing precise species identifications regardless of the taxonomic expertise of the investigator or the developmental stage of the specimens. However, the process is dependent on the completeness of the reference libraries. In this study, we sought to evaluate the potential of DNA metabarcoding to assess the seasonal (summer, autumn, and early spring) and spatial dynamics of zooplankton (four locations spanning ca. 6 km) in the Lima estuary (NW Portugal). Two genetic markers were used: the cytochrome c oxidase subunit I and the V4 hypervariable region of the ribosomal 18S rRNA genes. Overall, 327 species were recovered, and both markers displayed minute overlap (7% were detected with both markers). Species richness, composition, and taxonomic distinctness were majorly influenced by the season, with a declining tendency from summer (highest number of exclusive species, n = 74) to spring. Second to season, the taxa composition was influenced by spatial variation where the most downstream site displayed the highest number of exclusive species, n = 53. A total of 16 non-indigenous species were detected using metabarcoding, but only one (Austrominus modestus) has been documented out in the estuary. In conclusion, both the seasonal and spatial gradients influenced the recovered richness, composition, and taxonomic distinctness, confirming the great aptitude of DNA metabarcoding for providing higher density monitoring and shedding new light on the composition and dynamics of complex zooplankton communities.
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The World Health Organization identifies tuberculosis (TB), caused by Mycobacterium tuberculosis, as a leading infectious killer. Although conventional treatments for TB exist, they come with challenges such as a heavy pill regimen, prolonged treatment duration, and a strict schedule, leading to multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. The rise of MDR strains endangers future TB control. Despite these concerns, the hunt for an efficient treatment continues. One breakthrough has been the use of nanotechnology in medicines, presenting a novel approach for TB treatment. Nanocarriers, such as lipid nanoparticles, nanosuspensions, liposomes, and polymeric micelles, facilitate targeted delivery of anti-TB drugs. The benefits of nanocarriers include reduced drug doses, fewer side effects, improved drug solubility, better bioavailability, and improved patient compliance, speeding up recovery. Additionally, nanocarriers can be made even more targeted by linking them with ligands such as mannose or hyaluronic acid. This review explores these innovative TB treatments, including studies on nanocarriers containing anti-TB drugs and related patents.
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Pancreatic cancer represents one of the most lethal cancer types worldwide, with a 5-year survival rate of less than 5%. Due to the inability to diagnose it promptly and the lack of efficacy of existing treatments, research and development of innovative therapies and new diagnostics are crucial to increase the survival rate and decrease mortality. Nanomedicine has been gaining importance as an innovative approach for drug delivery and diagnosis, opening new horizons through the implementation of smart nanocarrier systems, which can deliver drugs to the specific tissue or organ at an optimal concentration, enhancing treatment efficacy and reducing systemic toxicity. Varied materials such as lipids, polymers, and inorganic materials have been used to obtain nanoparticles and develop innovative drug delivery systems for pancreatic cancer treatment. In this review, it is discussed the main scientific advances in pancreatic cancer treatment by nano-based drug delivery systems. The advantages and disadvantages of such delivery systems in pancreatic cancer treatment are also addressed. More importantly, the different types of nanocarriers and therapeutic strategies developed so far are scrutinized.
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ABSTRACT: Leukemia cutis corresponds to skin infiltration by malignant hematopoietic cells. It is most commonly reported in acute myeloid leukemia, particularly in subtypes with a monocytic component. Its clinical manifestations are extremely variable, and histopathologic diagnosis of cutaneous leukemic infiltrates may be challenging. We report the first case of cutaneous, that is, extramedullary, aleukemic relapse of acute myeloid leukemia within an unusual chilblain-like eruption that imposed a challenging clinical and histopathologic diagnosis. Primary chilblains are uncommon in the elderly, and a systemic underlying cause should be thoroughly investigated. In patients presenting with atypical chilblains (ie, persistent chilblains developing even without exposure to cold temperatures and/or refractory to therapy) and with a history of hematologic disorders such as leukemias, histopathologic examination is crucial to identify leukemic or aleukemic phases of relapse of underlying leukemia and initiate timely treatment.
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Pérnio , Exantema , Leucemia Mieloide Aguda , Neoplasias Cutâneas , Idoso , Humanos , Pérnio/diagnóstico , Temperatura BaixaRESUMO
Animal detection through DNA present in environmental samples (eDNA) is a valuable tool for detecting rare species, that are difficult to observe and monitor. eDNA-based tools are underpinned by molecular evolutionary principles, key to devising tools to efficiently single out a targeted species from an environmental sample. Here, we present a comprehensive review of the use of eDNA-based methods for the detection of targeted animal species, such as rare, endangered, or invasive species, through the analysis of 549 publications (2008-2022). Aquatic ecosystems have been the most surveyed, in particular, freshwaters (74 %), and to a less extent marine (14 %) and terrestrial systems (10 %). Vertebrates, in particular, fish (38 %), and endangered species, have been the focus of most of these studies, and Cytb and COI are the most employed markers. Among invertebrates, assays have been mainly designed for Mollusca and Crustacea species (21 %), in particular, to target invasive species, and COI the most employed marker. Targeted molecular approaches, in particular qPCR, have been the most adopted (75 %), while eDNA metabarcoding has been rarely used to target single or few species (approx. 6 %). However, less attention has been given in these studies to the effects of environmental factors on the amount of shed DNA, the differential amount of shed DNA among species, or the sensitivity of the markers developed, which may impact the design of the assays, particularly to warrant the required detection level and avoid false negatives and positives. The accuracy of the assays will also depend on the availability of genetic data and vouchered tissue or DNA samples from closely related species to assess both marker and primers' specificity. In addition, eDNA-based assays developed for a particular species may have to be refined for use in a new geographic area taking into account site-specific populations, as well as any intraspecific variation.
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DNA Ambiental , Ecossistema , Animais , Vertebrados/genética , Peixes/genética , DNA/genética , Espécies Introduzidas , Biodiversidade , Monitoramento Ambiental/métodosRESUMO
The determination of the number of plasmid copies in each cell of Lactococcus lactis is critical for the control and regulation of the production of recombinant proteins and plasmids. This protocol describes a method for the determination of the plasmid copy number per genome of L. lactis, which is based on the detection by real-time quantitative PCR of the number of plasmid molecules and the number of chromosomes and subsequently their ratio after calculating the amplification efficiency.
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Lactococcus lactis , Lactococcus lactis/genética , Variações do Número de Cópias de DNA , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Managing marine nonindigenous species (mNIS) is challenging, because marine environments are highly connected, allowing the dispersal of species across large spatial scales, including geopolitical borders. Cross-border inconsistencies in biosecurity management can promote the spread of mNIS across geopolitical borders, and incursions often go unnoticed or unreported. Collaborative surveillance programs can enhance the early detection of mNIS, when response may still be possible, and can foster capacity building around a common threat. Regional or international databases curated for mNIS can inform local monitoring programs and can foster real-time information exchange on mNIS of concern. When combined, local species reference libraries, publicly available mNIS databases, and predictive modeling can facilitate the development of biosecurity programs in regions lacking baseline data. Biosecurity programs should be practical, feasible, cost-effective, mainly focused on prevention and early detection, and be built on the collaboration and coordination of government, nongovernment organizations, stakeholders, and local citizens for a rapid response.
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Despite significant progress in unraveling the genetic causes of neurodevelopmental disorders (NDDs), a substantial proportion of individuals with NDDs remain without a genetic diagnosis after microarray and/or exome sequencing. Here, we aimed to assess the power of short-read genome sequencing (GS), complemented with long-read GS, to identify causal variants in participants with NDD from the National Institute for Health and Care Research (NIHR) BioResource project. Short-read GS was conducted on 692 individuals (489 affected and 203 unaffected relatives) from 465 families. Additionally, long-read GS was performed on five affected individuals who had structural variants (SVs) in technically challenging regions, had complex SVs, or required distal variant phasing. Causal variants were identified in 36% of affected individuals (177/489), and a further 23% (112/489) had a variant of uncertain significance after multiple rounds of re-analysis. Among all reported variants, 88% (333/380) were coding nuclear SNVs or insertions and deletions (indels), and the remainder were SVs, non-coding variants, and mitochondrial variants. Furthermore, long-read GS facilitated the resolution of challenging SVs and invalidated variants of difficult interpretation from short-read GS. This study demonstrates the value of short-read GS, complemented with long-read GS, in investigating the genetic causes of NDDs. GS provides a comprehensive and unbiased method of identifying all types of variants throughout the nuclear and mitochondrial genomes in individuals with NDD.
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Genoma Humano , Transtornos do Neurodesenvolvimento , Humanos , Genoma Humano/genética , Mapeamento Cromossômico , Sequência de Bases , Mutação INDEL , Transtornos do Neurodesenvolvimento/genéticaRESUMO
Cysteine residues can undergo multiple posttranslational modifications with diverse functional consequences, potentially behaving as tunable sensors. The intermediate filament protein vimentin has important implications in pathophysiology, including cancer progression, infection, and fibrosis, and maintains a close interplay with other cytoskeletal structures, such as actin filaments and microtubules. We previously showed that the single vimentin cysteine, C328, is a key target for oxidants and electrophiles. Here, we demonstrate that structurally diverse cysteine-reactive agents, including electrophilic mediators, oxidants and drug-related compounds, disrupt the vimentin network eliciting morphologically distinct reorganizations. As most of these agents display broad reactivity, we pinpointed the importance of C328 by confirming that local perturbations introduced through mutagenesis provoke structure-dependent vimentin rearrangements. Thus, GFP-vimentin wild type (wt) forms squiggles and short filaments in vimentin-deficient cells, the C328F, C328W, and C328H mutants generate diverse filamentous assemblies, and the C328A and C328D constructs fail to elongate yielding dots. Remarkably, vimentin C328H structures resemble the wt, but are strongly resistant to electrophile-elicited disruption. Therefore, the C328H mutant allows elucidating whether cysteine-dependent vimentin reorganization influences other cellular responses to reactive agents. Electrophiles such as 1,4-dinitro-1H-imidazole and 4-hydroxynonenal induce robust actin stress fibers in cells expressing vimentin wt. Strikingly, under these conditions, vimentin C328H expression blunts electrophile-elicited stress fiber formation, apparently acting upstream of RhoA. Analysis of additional vimentin C328 mutants shows that electrophile-sensitive and assembly-defective vimentin variants permit induction of stress fibers by reactive species, whereas electrophile-resistant filamentous vimentin structures prevent it. Together, our results suggest that vimentin acts as a break for actin stress fibers formation, which would be released by C328-aided disruption, thus allowing full actin remodeling in response to oxidants and electrophiles. These observations postulate C328 as a "sensor" transducing structurally diverse modifications into fine-tuned vimentin network rearrangements, and a gatekeeper for certain electrophiles in the interplay with actin.
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Actinas , Filamentos Intermediários , Filamentos Intermediários/química , Actinas/genética , Actinas/química , Vimentina/genética , Vimentina/química , Cisteína/metabolismo , Oxidantes/metabolismoRESUMO
Background: Idiopathic inflammatory myopathies (IIM) are a rare heterogeneous group of diseases characterised by chronic skeletal muscle inflammation, but other organs are also frequently involved. IMM represent a diagnostic challenge and a multidisciplinary approach is important to ensure successful diagnosis and adequate follow-up of these patients. Objective: To describe the general functioning of our multidisciplinary myositis clinic, highlighting the benefits of multidisciplinary team management in patients with confirmed or suspected IIM and to characterise our clinical experience. Methods: Description of the organization of a dedicated multidisciplinary myositis outpatient clinic, supported by IMM specific electronic assessment tools and protocols based on our Portuguese Register - Reuma.pt. In addition, an overview of our activity between 2017 and 2022 is provided. Results: An IIM multidisciplinary care clinic, based on a close collaboration between Rheumatologists, Dermatologists and Physiatrist is detailed in this paper. One hundred and eighty-five patients were assessed in our myositis clinic; 138 (75%) of those were female, with a median age of 58 [45-70] years. At the last appointment, 130 patients had a confirmed IIM diagnosis, and the mean disease duration was 4 [2-6] years. The most frequent diagnosis was dermatomyositis (n = 34, 26.2%), followed by antisynthetase syndrome (n = 27, 20.8%) and clinically amyopathic/paucimyopathic dermatomyositis (n = 18, 13.8%). Twenty-four patients (18.5%) were on monotherapy and 94 (72.3%) were on combination therapy. Conclusion: A multidisciplinary approach is important to ensure the correct diagnosis and follow-up of these patients. A myositis clinic, with a standardised practice at a tertiary hospital level, contributes to a standardization of care and opens research opportunities.
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Rice is the second most important cereal crop and is vital for the diet of billions of people. However, its consumption can increase human exposure to chemical contaminants, namely mycotoxins and metalloids. Our goal was to evaluate the occurrence and human exposure of aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEN), and inorganic arsenic (InAs) in 36 rice samples produced and commercialized in Portugal and evaluate their correlation. The analysis of mycotoxins involved ELISA, with limits of detection (LODs) of 0.8, 1 and 1.75 µg kg-1 for OTA, AFB1, and ZEN, respectively. InAs analysis was carried out by inductively coupled plasma mass spectrometry (ICP-MS; LOD = 3.3 µg kg-1). No sample showed contamination by OTA. AFB1 was present in 2 (4.8%) samples (1.96 and 2.20 µg kg-1), doubling the European maximum permitted level (MPL). Concerning ZEN, 88.89% of the rice samples presented levels above the LOD up to 14.25 µg kg-1 (average of 2.75 µg kg-1). Regarding InAs, every sample presented concentration values above the LOD up to 100.0 µg kg-1 (average of 35.3 µg kg-1), although none surpassed the MPL (200 µg kg-1). No correlation was observed between mycotoxins and InAs contamination. As for human exposure, only AFB1 surpassed the provisional maximum tolerable daily intake. Children were recognized as the most susceptible group.
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Micotoxinas , Oryza , Zearalenona , Criança , Humanos , Micotoxinas/análise , Oryza/química , Projetos Piloto , Portugal , Contaminação de Alimentos/análise , Zearalenona/análise , Aflatoxina B1/análise , Medição de RiscoRESUMO
Background: Viral or bacterial infections can trigger auto-immune inflammatory reactions and conditions in children. Self-reactivity arises due to similarities in molecular structures between pathogenic microorganisms and regular body structures with consequent immune-cross reactions. Reactivation of latent Varicella Zoster Virus (VZV) infections can cause neurological sequalae, including cerebellitis, post-herpetic neuralgias, meningo/encephalitis, vasculopathy and myelopathy. We propose a syndrome caused by auto-immune reactivity triggered by molecular mimicry between VZV and the brain, culminating in a post-infectious psychiatric syndrome with childhood VZV infections. Case presentation: Two individuals, a 6-year-old male and 10-year-old female developed a neuro-psychiatric syndrome 3-6 weeks following a confirmed VZV infection with intrathecal oligoclonal bands. The 6-year-old male presented with a myasthenic syndrome, behavior deterioration and regression in school, he was poorly responsive to IVIG and risperidone, however had a pronounced response to steroid treatment. The 10-year-old female presented with marked insomnia, agitation, and behavioral regression as well as mild bradykinesia. A trial of neuroleptics and sedatives resulted in a mild unsustained reduction in psychomotor agitation and IVIG was also unsuccessful, however the patient was very responsive to steroid therapy. Conclusion: Psychiatric syndromes with evidence of intrathecal inflammation temporally related to VZV infections that are responsive to immune modulation have not been described before. Here we report two cases demonstrating neuro-psychiatric symptoms following VZV infection, with evidence of persistent CNS inflammation following the resolution of infection, and response to immune modulation.
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The poultry industry, in order to prevent and control coccidiosis caused by Eimeria spp., widely uses coccidiostats as feed additives. The main objective of this study was to evaluate the presence of nine coccidiostats in 62 egg samples by UHPLC-MS/MS. Overall, detection frequency and average concentration were 90.3% (56/62) and 106.3 µg kg-1, respectively. Only diclazuril and nicarbazin were detected. Diclazuril, only found in home-raised eggs, showed an overall detection frequency of 8.1% (5/62), with average and maximum concentrations of 0.46 ± 1.90 µg kg-1 and 13.6 µg kg-1, respectively. Nicarbazin presented an overall higher frequency, 88.7% (55/62), with levels up to 744.8 µg kg-1. Additionally, four samples (6.5%) presented both nicarbazin and diclazuril. Home-raised egg samples (n = 28) showed a detection frequency of 89.3%, with nicarbazin found in more samples (85.7% vs. 17.9%) and at higher levels (266.3 ± 169.4 µg kg-1 vs. 0.91 ± 2.78 µg kg-1) when compared to diclazuril. In supermarket samples (n = 34), only nicarbazin was detected in 31 samples (91.1%), with an average of 167.6 ± 62.2 µg kg-1. Considering the average contamination scenario, consumers' health should not be adversely affected by egg consumption. In every scenario considered, children were the most vulnerable population group.
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The Azorean dairy industry is based on a semi-intensive model. Due to the few storage facilities and the large climatic fluctuations that characterize the Azores, the complete feed mixture (total mixed ration - TMR) is susceptible to the proliferation of a variety of fungi and mycotoxin contamination. Thus, chronic ingestion of these xenobiotics may lead to increased susceptibility to disease, loss of reproductive performance and, in the case of dairy cattle, decreased productivity and quality of milk produced. Since it is impossible to eliminate completely the presence of mycotoxins, it is essential to ensure the implementation of strategies to reduce their concentration in products intended for food and feed, as well as to monitor and control the levels present in food. This study aimed to evaluate the occurrence zearalenone (ZEA) in feed (TMR) in four dairy cattle farms on the Azorean Island of São Miguel and relate it to the occurrence of this mycotoxin in the milk produced, by associating several production and health indicators translated by the monthly milk contrast. To this end, the monthly milk contrast data, the determination of ZEA in the ingested feed and the (individual) milk samples from the four farms included in the study were used. Eighty-three (98.8%) of the milk samples under study had detectable ZEA levels (1.56 ± 1.36 µg/L), higher than reported in previous similar studies. Although ZEA concentration was not significantly associated with any production indicator analyzed (days in milk, age at calving, milk yield, protein content, butyrate content, somatic cell concentration and urea), it was found that the production regime and type of feed management is a major factor in the exposure of animals to high mycotoxin contents. Further studies are recommended to ensure continued monitoring and reduction of the risk associated with exposure of animals and humans to ZEA.