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ABSTRACT Aflatoxins are secondary metabolites toxic to human and animal health produced by Aspergillus flavus, and which contaminate food throughout the production chain. Knowing and characterizing the fungal population present in food helps us to estimate risk and design measures to mitigate fungal contamination. Thus, the objective of this work was characterizing the toxigenic potential of Aspergillus isolates from corn in in vitro models. For this purpose, two isolates of Aspergillus flavus (CCM-AS02, CCM-AS29) and one of Aspergillus luchuensis (CCM-AS04) from the CCM-UNA culture collection were used. To evaluate the production of aflatoxins in synthetic culture medium, the isolates were plated on coconut agar and yeast extract agar, and the presence of fluorescence was evaluated under UV light (A= 360 nm). To determine the type of aflatoxin, a thin layer chromatography was performed. Finally, an in vitro infection was carried out with the isolates under study, from avatí-morotí maize, acquired commercially, and the aflatoxin concentration was determined with the Afla-V®-VICAM® lateral fluid rapid immunoassay kit. With the results obtained, it can be concluded that the isolates of A. flavus CCM-AS02 and CCM-AS29 from corn are aflatoxigenic under environmental conditions that simulate natural ones and that coincide with the predominant ones in our country, therefore, it is necessary to create awareness of the risk posed by the contamination of corn grains with Aspergillus and aflatoxins in our region and the need to take preventive measures to control this fungus.

RESUMEN Las aflatoxinas son metabolitos secundarios tóxicos para salud humana y animal producidos por Aspergillus flavus, y que contaminan a los alimentos a lo largo de la cadena productiva. Conocer y caracterizar la población fúngica presente en los alimentos nos sirve para estimar riesgo y diseñar medidas para mitigarlo. Siendo así, el objetivo de este trabajo es caracterizar el potencial toxigénico de aislados de Aspergillus provenientes de maíz en modelos in vitro. Para tal fin, se utilizaron dos aislados de A. flavus (CCM-AS02, CCM-AS29) y uno de Aspergillus luchuensis (CCM-AS04) de la colección de cultivos CCM-UNA. Para evaluar la producción de aflatoxinas en medio de cultivo sintético, se sembraron los aislados en agar coco y agar extracto de levadura y se evaluó la presencia de fluorescencia bajo luz UV (A= 360 nm). Para determinar el tipo de aflatoxina, se realizó cromatografía en capa delgada. Por último, se realizó la infección in vitro con los aislados en estudio, de maíz avatí-morotí, adquirido comercialmente y se determinó la concentración de aflatoxinas con la prueba de inmunoensayo rápido de flujo lateral Afla - V®-VICAM®. Con los resultados obtenidos se puede concluir que los aislados de A. flavus CCM-AS02 y CCM-AS29 provenientes de maíz son aflatoxigénicos en las condiciones ambientales que simulan las naturales y que coinciden con las predominantes en nuestro país, por ello, es necesario crear conciencia del riesgo que representa la contaminación de los granos de maíz con Aspergillus y aflatoxinas y la necesidad de tomar medidas preventivas de control de este hongo.

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ABSTRACT: The presence of aflatoxin M1 (AFM1) in milk is a public health concern because milk is a significant part of human diets worldwide. In economies where AFM1 monitoring is low or nonexistent, the possibility of AFM1 contamination might be increased. Our study was conducted to detect and quantify AFM1 in fluid milk and milk drinks of various brands, fat concentrations, packages, and heat treatments sold in the Metropolitan Area of Asunción, Paraguay. Eighty samples were collected from supermarkets in the Metropolitan Area of Asunción following a nonprobability sampling method. An enzyme-linked immunosorbent assay for AFM1 (25 to 500 ppt) was used to quantify the toxin, and results were analyzed with nonparametric methods. All samples were positive values for AFM1 (above the detection limit of 25 ng/kg); 85% of the samples had 30 to 50 ng/kg, and 15% had >500 ng/kg. No significant difference in AFM1 concentration was found based on fat concentration, heat treatment, or type of packaging of these milk products; however, significant differences were found between brands.

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The genus Pyricularia contains several fungal species known to cause diseases on plants in the Poaceae family (Klaubauf et al. 2014; Wang et al. 2019). While sampling for P. oryzae during March-2015 and April-2018, common weed Cenchrus echinatus L. was observed with leaf lesions in and around experimental wheat fields in the departments of Canindeyú and Itapúa. C. echinatus samples from both locations displayed similar leaf lesions, varying from small light brown pinpoint to elliptical brown lesions with greyish center. Symptomatic leaves were surface disinfested and cultured on potato dextrose agar (PDA) amended with 1% gentamicin at 25°C. Two monosporic isolates were obtained, one from Itapúa (ITCeh117) and the other from Canindeyú (YCeh55). The isolates were subsequently grown on oatmeal agar (OA) and PDA under a 12-h photoperiod at 25°C and evaluated after ten days for colony diameter, sporulation, macroscopic and microscopic features. Colonies on OA reached up to 4.8 cm diameter and were light grey, whereas colonies on PDA reached up to 5.3 cm diameter and were brown with grey centers, with cottony mycelium and broad white rims. Mycelium consisted of smooth, hyaline, branched, septate hyphae 4-4.5 µm diameter. Conidiophores were erect, straight or curved, unbranched, medium brown and smooth. Conidia were solitary, pyriform, pale brown, smooth, granular, 2-septate, 32-33 × 9-10 µm; truncated with protruding hilum and varied in length from 1.0 to 1.5 µm and diameters from 2.0 to 2.2 µm. Both isolates were similar and identified as Pyricularia pennisetigena, according to morphological and morphometric characteristics (Klaubauf et al. 2014). Subsequently, genomic DNA was extracted from each isolate using the primers described in Klaubauf et al. (2014) to amplify and sequence the internal transcribed spacers (ITS), partial large subunit (LSU), partial RNA polymerase II large subunit gene (RPB1), partial actin gene (ACT), and partial calmodulin gene (CAL). Sequences from each isolate (YCeh55/ITCeh117) were deposited in GenBank with the following submission ID for ITS: MN947521/MN947526, RPB1: MN984710/MN984715, LSU: MN944829/MN944834, ACT: MN917177/MN917182, and CAL: MN984688/MN984693. Phylogenetic analysis was conducted using the software Beast v1.10.4. The results obtained from the concatenated matrix of the five loci placed these isolates in the P. pennisetigena clade. To confirm pathogenicity, each isolate was adjusted to 5×104 conidia/ml of sterile water and C. echinatus plants were sprayed with the conidial suspension for isolate YCeh55, ITCeh117 or sterile water using an oilless airbrush sprayer until runoff. The three treatments were kept in the greenhouse at 25-28°C and about 75% relative humidity under natural daylight. Each treatment included three to five inoculated plants and 10 leaves were evaluated per treatment. Symptoms were observed 8-15 days after inoculation and were similar to those originally observed in the field for both isolates, whereas the control plants remained asymptomatic. P. pennisetigena was re-isolated from the inoculated leaves fulfilling Koch's postulates. To our knowledge, this is the first report of leaf blight on C. echinatus caused by P. pennisetigena in Paraguay. The occurrence of P. pennisetigena in the region and its ability to infect economically important crops such as wheat and barley (Klaubauf et al. 2014; Reges et al., 2016, 2018) pose a potential threat to agriculture in Paraguay.

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RESUMEN Introducción: La aflatoxina M1 (AFM1) es un metabolito tóxico derivado de la aflatoxina B. Su ingestión en lactantes se ha relacionado con retraso en el crecimiento, aumento de susceptibilidad a enfermedades infecciosas, reducción de la eficiencia en la inmunización y cirrosis. En Paraguay se recomienda lactancia exclusiva hasta los 6 meses, sin embargo se comercializan diferentes marcas de fórmulas infantiles dirigidas a lactantes menores de 6 meses. Objetivo: Detectar y cuantificar la presencia de AFM1 en fórmulas para lactantes comercializadas en el Área Metropolitana. Materiales y Métodos: Se adquirieron fórmulas fluidas (n=18) y en polvo (n=91) para lactantes de 0 a 12 meses de farmacias y supermercados del Área Metropolitana de Asunción y fueron analizados mediante el ensayo de inmunoafinidad ligado a enzimas (ELISA). Resultados: 9,75% (0 a 6 meses) y 2% (6 a 12 meses) de las fórmulas lácteas en polvo y 100% de las fórmulas fluidas resultaron positivas para AFM1. La mediana de contenido de AFM1 en formulas en polvo fue de 1820 ng/kg y 510 ng/kg en las marcas A y B. En las fórmulas líquidas fue de 31,8 ng/kg y 33,6 ng/kg para las dos marcas analizadas respectivamente, p=0,0001. Conclusiones: Se detectó AFM1 en todas las fórmulas líquidas analizadas, y en el 2 y 9,7% de las fórmulas en polvo de las marcas Ay B respectivamente Los niveles de AFM1 fueron mayores en las fórmulas en polvo.

ABSTRACT Introduction: Aflatoxin M1 (AFM1) is a toxic metabolite derived from aflatoxin B. Its ingestion in infants has been related to growth retardation, increased susceptibility to infectious diseases, reduced immunization efficiency, and cirrhosis. In Paraguay, exclusive breastfeeding is recommended up to 6 months of age, however, different brands of infant formulas targeted at this age range are marketed. Objective: To detect and quantify the presence of AFM1 in infant formulas marketed in the Metropolitan Area. Materials and Methods: Fluid (n = 18) and powder (n = 91) formulas for infants aged 0 to 12 months were purchased from pharmacies and supermarkets in the Metropolitan Area of Asunción and were analyzed using the enzyme-linked immunoaffinity assay (ELISA). Results: 9.75% (0 to 6 months) and 2% (6 to 12 months) of the powdered milk formulas and 100% of the fluid formulas were positive for AFM1. The median content of AFM1 in powder formulas was 1820 ng/kg and 510 ng/kg in brands A and B, while in liquid formulas it was 31.8 ng/kg and 33.6 ng/kg for the two brands analyzed respectively (p = 0.0001). Conclusions: AFM1 was detected in all the liquid formulas analyzed, and in 2 and 9.7% of the powder formulas of brands A and B, respectively. The levels of AFM1 were higher in the powder formulas.

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Alcoholic beverages can be contaminated with mycotoxins. Ochratoxin A (OTA) is the most frequently detected mycotoxinin wine and is produced by several species of Aspergillus. This mycotoxin is nephrotoxic and carcinogenic. In beer, the most commonly identified mycotoxin is deoxynivalenol (DON). Ingestion of food contaminated with DON has been associated with adverse gastrointestinal effects. Despite the harmful effects of mycotoxins on health, there are no regulations regarding their limits in alcoholic beverages in Paraguay. Here we determine the presence of OTA and DON in wine and beer, respectively. Four commercial brands of wine and twenty-nine brands of craft and industrial beerwere tested by the Agra quant ELISA method. One brand of wine was positive for OTA and seven brands of beer (one of them craft) were positive for DON. The values found for both toxins are below the recommended maximum intake proposed by international standards. Giving the high consumption of these products in the country, regulations and monitoring systems mustbe established to check the maximum levels of mycotoxins allowed in alcoholic beverages.

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Introducción: Las micotoxicosis son enfermedades producidas por micotoxinas, metabolitos secundarios tóxicos producidos por hongos filamentosos. Los lactantes son especialmente susceptibles a este tipo de toxinas debido a la inmadurez anatómica y funcional de sus sistemas digestivo e inmune, lo que se refleja en la relación entre la cantidad de alimento ingerida y su peso. Objetivo: Determinar la presencia de micotoxinas en alimentos para lactantes comercializados en farmacias y supermercados del Área Metropolitana. Materiales y métodos: Se colectaron al azar 66 unidades de productos de seis marcas diferentes de preparados y colados comerciales importados, dulces y salados, de farmacias y supermercados del Área Metropolitana. Posteriormente, fueron analizados mediante el ensayo de inmunoafinidad ligado a enzimas (ELISA). Se realizó el análisis de varianza y la posterior comparación de medias de las concentraciones de micotoxinas mediante la prueba de Tukey (IC= 95%), con el estadístico InfoStat®. Resultados: Las micotoxinas prevalentes fueron aflatoxinas (AF) y Toxina T2, que se presentaron en 39% de las unidades muestrales analizadas, tanto en preparados como en colados. En tercer orden de importancia se encuentra ocratoxina A (OTA), detectada en 18% de las mismas. En cuarto lugar, deoxinivalenol (DON) se detectó en 4% los productos. Se presentaron diferencias significativas entre los preparados y los colados, siendo los colados los que en media presentaron niveles más altos de concentraciones de todas las micotoxinas estudiadas. Conclusiones: Se constataron niveles variables de AF, OTA, T2 y DON en los alimentos para lactantes comercializados en el Área Metropolitana. Los colados presentaron concentraciones más elevadas de micotoxinas en media en todos los productos analizados. Tanto AF como OTA superaron los límites máximos permitidos por las normas internacionales.

Introduction: Mycotoxicoses are diseases caused by mycotoxins, secondary toxic metabolites produced by filamentous fungi. Infants are especially susceptible to this type of toxins due to the anatomical and functional immaturity of their digestive and immune systems, which is related to the amount of food eaten and their weight. Objective: To determine the presence of mycotoxins in foods for infants sold in pharmacies and supermarkets in the Metropolitan Area. Materials and methods: 66 units of products from six different brands of imported commercial sweet and salty preparations and strained foods from pharmacies and supermarkets in the Metropolitan Area were randomly collected. Subsequently, they were analyzed by the enzyme-linked immunoaffinity test (ELISA). The variance analysis and the subsequent comparison of means of mycotoxin concentrations were performed using the Tukey test (95% CI), with the InfoStat® statistic. Results: The prevalent mycotoxins were aflatoxins (AF) and T-2 Toxin, which were present in 39% of the sample units analyzed, both in preparations and in strains. In third order of importance we detected Ochratoxin A (OTA) in 18% of the units. Fourth, deoxinivalenol (DON) products were detected in 4%. There were significant differences between the preparations and the strains, with the strains having the highest concentration levels of all the mycotoxins studied. Conclusions: Variable levels of AF, OTA, T-2 and DON were found in infant foods marketed in the Metropolitan Area. The strains showed a higher average concentrations of mycotoxins in all the products analyzed. Both AF and OTA exceeded the maximum limits allowed by international standards.