Paula Rodrigues

A polyphasic approach to the identification of aflatoxigenic and non-aflatoxigenic strains of Aspergillus Section Flavi isolated from Portuguese almonds

A polyphasic approach consisting of morphological, chemical and molecular characterization was applied to 31 isolates of Aspergillus Section Flavi originating from Portuguese almonds, with the aim of characterizing and identifying aflatoxigenic and non-aflatoxigenic strains. On the basis of morphological characters (mainly colony color on Czapek-Dox agar and conidia morphology), we found two distinct groups among the population under study: 18 isolates (58%) had dark-green colonies and rough conidia, and were classified as Aspergillus parasiticus; the remaining 13 isolates (42%) had yellow-green colonies and smooth to finely rough globose conidia, and were classified as Aspergillus flavus. Chemical characterization involved the screening of the isolates for aflatoxins B (AFB) and G (AFG), and also for cyclopiazonic acid (CPA), by HPLC with fluorescence and UV detection, respectively. All A. parasiticus isolates were strong AFB and AFG producers, but no CPA production was detected, showing a consistent mycotoxigenic pattern. The A. flavus isolates showed to be more diversified, with 77% being atoxigenic, whereas 15% produced CPA and low levels of AFB and 8% produced the 3 groups of mycotoxins. Aflatoxin production was also screened on Coconut Agar Medium (CAM), and the results were consistent with the HPLC analysis. Sclerotia production showed no correlation to aflatoxigenicity. Molecularly, two genes of the aflatoxin biosynthetic pathway, aflD (=nor1) and aflQ (=ord1=ordA) were tested for presence and expression (by PCR and RT-PCR, respectively). The presence of both genes did not correlate with aflatoxigenicity. aflD expression was not considered a good marker for differentiating aflatoxigenic from non-aflatoxigenic isolates, but aflQ showed a good correlation between expression and aflatoxin-production ability.

Physicochemical, microbiological and antimicrobial properties of commercial honeys from Portugal.

The present study aimed to characterize five commercial honeys available in the Portuguese market in respect to their floral origins, physicochemical parameters and microbial safety and commercial quality assessment. Pollen profile, colour, moisture content, ash, acidity, electrical conductivity, pH, reducing sugars, apparent sucrose and HMF were the parameters analysed in each honey sample. Aerobic mesophiles, moulds and yeasts, fecal coliforms and sulphite-reducing clostridia were the microbial contaminants of interest studied. The antimicrobial effect against four fermentative yeasts was determined. Concerning the physicochemical parameters, all honey samples were found to meet European Legislation (EC Directive 2001/110) for all parameters, except for HMF and apparent sucrose. Microbiologically, the commercial quality was considered good and all samples showed to be negative in respect to safety parameters. We also verified that the presence of honey differentially affected the growth of fermentative yeasts under study, depending on the type of yeast, but this growth was not significantly influenced by the type of honey used.

Aflatoxigenic fungi and aflatoxins in Portuguese almonds

Aflatoxin contamination of nuts is an increasing concern to the consumers health. Portugal is a big producer of almonds, but there is no scientific knowledge on the safety of those nuts, in terms of mycotoxins. The aim of this paper was to study the incidence of aflatoxigenic fungi and aflatoxin contamination of 21 samples of Portuguese almonds, and its evolution throughout the various stages of production. All fungi belonging to Aspergillus section Flavi were identified and tested for their aflatoxigenic ability. Almond samples were tested for aflatoxin contamination by HPLC-fluorescence. In total, 352 fungi belonging to Aspergillus section Flavi were isolated from Portuguese almonds: 127 were identified as A. flavus (of which 28% produced aflatoxins B), 196 as typical or atypical A. parasiticus (all producing aflatoxins B and G), and 29 as A. tamarii (all nonaflatoxigenic). Aflatoxins were detected in only one sample at 4.97 μg/kg.

Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecular approaches.

Aims:  Section Flavi is one of the most significant sections in the genus Aspergillus. Taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. This work aimed to identify isolates from section Flavi by combining various classic phenotypic and genotypic methods as well as the novel approach based on spectral analysis by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF ICMS) and to evaluate the discriminatory power of the various approaches in species identification.

Three new species of Aspergillus section Flavi isolated from almonds and maize in Portugal.

Three new aflatoxin-producing species belonging to Aspergillus section Flavi are described. They are Aspergillus mottae, A. sergii and A. transmontanensis. These species were isolated from Portuguese almonds and maize. An investigation examined morphology, extrolite production and DNA sequence data to characterize these isolates and describe the new species. Phylogenetic analysis showed that A. transmontanensis and A. sergii form a clade with A. parasiticus whereas A. mottae shares a most recent common ancestor with the combined A. flavus and A. parasiticus clade.

Prevalence of Babesia microti-like infection in red foxes (Vulpes vulpes) from Portugal

The prevalence of piroplasm (order Piroplasmida) infection was assessed in blood and bone marrow samples from 91 red foxes (Vulpes vulpes) from northern, central and southern Portugal by means of molecular methods. PCR for the 18S rRNA gene of Babesia spp. followed by sequencing revealed 63 foxes positive for the Babesia microti-like piroplasm (syn. Theileria annae) (69.2%; 95% confidence interval [CI]: 58.7-78.5%) and one fox positive for Babesia canis (1.1%; 95% CI: 0.0-6.0%). Positivity to the B. microti-like piroplasm or B. canis in 43 blood samples (83.7%) was significantly higher (p<0.001) than in 43 paired bone marrow samples (20.9%). There were no statistically significant differences in the prevalence of infection between genders (p=0.219) or age groups (<2 years vs. ≥ 2 years) (p=1.0). This is the first report of the B. microti-like piroplasm in foxes from Portugal as well as the first report on detection by PCR and genotyping of B. canis in a red fox worldwide. A natural cycle of the B. microti-like piroplasm is suggested in red fox populations based on the high prevalence of the protozoan. Red foxes might be a reservoir of the B. microti-like piroplasm and a source of infection to dogs.

HPLC method for simultaneous detection of aflatoxins and cyclopiazonic acid

Aspergillus species in section Flavi are among the most relevant mycotoxigenic fungi. The organisms are well-known producers of the highly carcinogenic aflatoxins and of other mycotoxins, such as cyclopiazonic acid. Aflatoxins and cyclopiazonic acid analyses can be routinely used for identification purposes within the section. Two separate chromatographic runs with distinct columns and detectors for each toxin were required in previous reports. A straightforward high performance liquid chromatography (HPLC) procedure for the simultaneous detection of these compounds in fungal cultures was developed in the present work using a methanol/water mobile phase, postcolumn photochemical derivatisation and fluorescence detection. The proposed method was tested with standards and fungal extracts of 24 Aspergillus section Flavi strains and compared to the common individual detection of these mycotoxins by HPLC analyses.

Opisthorchis felineus in cat: case report

Opisthorchis felineus foi encontrado em um gato admitido no Hospital Veterinario da Universidade de Tras-os-Montes e Alto Douro. No momento de admissao, o animal apresentava-se em estado de choque, com hipotermia, desidratacao e mucosas palidas. As analises hematologicas revelaram linfopenia, eosinofilia e anemia moderada. A necropsia observou-se distensao da vesicula biliar e intensa infestacao parasitaria no figado. O estudo morfologico do parasita revelou tratar-se de Opisthorchis felineus.

Aspergillus westerdijkiae as a major ochratoxin A risk in dry-cured ham based-media

Penicillium nordicum is well known for its ability to produce high amounts of ochratoxin A (OTA) in cured meat-derived products. On the other hand, Aspergillus westerdijkiae, one of the most relevant OTA-producing species of the genus Aspergillus, is usually considered a major risk in carbon-rich food matrices of plant origin. The objective of this work was to evaluate, for the first time, the ecophysiological conditions governing growth, OTA production and sporulation of A. westerdijkiae (the type-strain and one ham-native strain), in comparison with P. nordicum, in dry-cured ham based medium. For that purpose, the interaction between temperature (15, 20, 25 and 30°C) and water activity (aw) (0.99, 0.97, 0.93, 0.90 and 0.85), achieved with a combination of ionic (NaCl) and non-ionic (glycerol) solutes, was studied by using dry-cured ham-based medium as a model system. Different OTA production profiles were found between the two genera, and also between the two strains of A. westerdijkiae, mostly in terms of amounts of OTA produced. The optimal OTA production conditions for A. westerdijkiae were at 0.94-0.97 aw and 20-25°C, and for P. nordicum at 0.95-0.97 aw between 18 and 22°C. Under these conditions, A. westerdijkiae produced 1934ng/g agar, while P. nordicum produced 712ng/g. None of the strains was able to produce detectable amounts of OTA at 0.85 aw, under all temperatures tested. Growth and sporulation were not good indicators of OTA production by A. westerdijkiae or P. nordicum. The results obtained show that A. westerdijkiae may represent a great potential risk of OTA contamination in dry-cured ham due to the high production under a wide range of conditions. Knowledge of the ecophysiology of important Aspergillus and Penicillium species and of their adaptability to the matrices can be determinant to adopt appropriate technological modifications during ham ripening process.

Incidence and diversity of the fungal genera Aspergillus and Penicillium in Portuguese almonds and chestnuts

Almonds (Prunus dulcis (Miller) D.A. Webb) and European (sweet) chestnuts (Castanea sativa Miller) are of great economic and social impact in Mediterranean countries, and in some areas they constitute the main income of rural populations. Despite all efforts to control fungal contamination, toxigenic fungi are ubiquitous in nature and occur regularly in worldwide food supplies, and these nuts are no exception. This work aimed to provide knowledge on the general mycobiota of Portuguese almonds and chestnuts, and its evolution from field to the end of storage. For this matter, 45 field chestnut samples and 36 almond samples (30 field samples and six storage samples) were collected in Trás-os-Montes, Portugal. All fungi belonging to genus Aspergillus were isolated and identified to the section level. Fungi representative of other genera were identified to the genus level. In the field, chestnuts were mainly contaminated with the genera Fusarium, Cladosporium, Alternaria and Penicillium, and the genus Aspergillus was only rarely found, whereas almonds were more contaminated with Aspergillus. In almonds, Aspergillus incidence increased significantly from field to the end of storage, but diversity decreased, with potentially toxigenic isolates belonging to sections Flavi and Nigri becoming more significant and widespread throughout storage. These fungi were determined to be moderately associated, which can be indicative of mycotoxin co-contamination problems if adequate storage conditions are not secured.