Daniele Sartori

Identification of fungi of the genus Aspergillus section nigri using polyphasic taxonomy

In spite of the taxonomy of the Aspergillus species of the Nigri Section being regarded as troublesome, a number of methods have been proposed to aid in the classification of this Section. This work aimed to distinguish Aspergillus species of the Nigri Section from foods, grains and caves on the basis in Polyphasic Taxonomy by utilizing morphologic and physiologic characters, and sequencing of s-tubulin and calmodulin genes. The morphologic identification proved useful for some species, such as A. carbonarius and Aspergillus sp UFLA DCA 01, despite not having been totally effective in elucidating species related to A. niger. The isolation of the species of the Nigri Section on Creatine Sucrose Agar (CREA) enabled to distinguish the Aspergillus sp species, which was characterized by the lack of sporulation and by the production of sclerotia. Scanning Electron microscopy (SEM) allowed distinguishing the species into two distinct groups. The production of Ochratoxin A (OTA) was only found in the A. carbonarius and A. niger species. The sequencing of β-tubulin gene was efficient in differing most of the Aspergillus species from the Nigri Section with the exception of Aspergillus UFLA DCA 01, which could not be distinguished from A. costaricaensis. This species is morphologically similar to A. costaricaencis for its low sporulation capacity and high sclerotia production, but it differs morphologically from A. costaricaensis for its conidial ornamentation and size of vesicles. Equally, based on partial calmodulin gene sequence data Aspergillus UFLA DCA 01 differs from A. costaricaensis.

Activity of selenium on cell proliferation, cytotoxicity, and apoptosis and on the expression of CASP9, BCL-XL and APC in intestinal adenocarcinoma cells

Intestinal cancers are correlated with diet. Thus, determining and understanding nutrient-genome interactions is important. The present work assessed the action of the oligoelement selenium on cell proliferation, cytotoxicity, and in situ apoptosis induction and on the expression CASP9, BCL-XL and APC genes in intestinal adenocarcinoma cells (HT29). HT29 cells were cultured and treated with selenium at concentrations of 5, 50 and 500ng/mL with or without the damage-inducing agent doxorubicin. These cells were then evaluated for cytotoxicity (MTT), cell proliferation and in situ apoptosis induction. To evaluate gene expression, only the cells treated with 500ng/mL of selenium were used. RNA was extracted from these cells, and the expressions of CASP9, BCL-XL and APC were analyzed by the RT-PCR method. The GAPDH gene was used as a reference gene. The MTT assay showed that selenium was not cytotoxic at any of the concentrations tested. The cell proliferation assay showed that selenium did not interfere with cell proliferation at the three concentrations tested. In contrast, when the three concentrations were combined with doxorubicin, a significant decrease in the proliferation rate was observed. The apoptosis rate was significantly increased in the selenium (500ng/mL) and doxorubicin group. CASP9 expression was increased and BCL-XL expression decreased in the selenium (500ng/mL) and doxorubicin group. APC was significantly increased in the selenium group alone. These results show that selenium increases apoptosis, especially when it is associated with a damage-inducing agent. Also, selenium has an important role in the expression of the APC gene, which is related to cell cycle regulation.

The biodiversity of Aspergillus section Flavi in brazil nuts: From rainforest to consumer

A total of 288 brazil nut samples (173 kernel and 115 shell) from the Amazon rainforest region and São Paulo State, Brazil were collected at different stages of brazil nut production. Samples were analysed for: percentages of aflatoxigenic fungal species and potential for aflatoxin production and presence of aflatoxins. Aspergillus nomius was the most common species found (1235 isolates) which amounted to 30% of the total species with potential to produce aflatoxins. This species is of concern since 100% of all isolates produced aflatoxins B(1), B(2), G(1) and G(2). Aspergillus flavus was almost equally common (1212 isolates) although only 46% produced aflatoxins under laboratory conditions, and only aflatoxins B(1) and B(2). Low number of other species with the potential to produce aflatoxins was isolated: Aspergillus arachidicola and Aspergillus bombycis produced B and G aflatoxins whilst Aspergillus pseudotamarii produced only aflatoxin B(1). The total aflatoxin levels found in samples taken from the rainforests was 0.7 μg/kg, from processing plants before and after sorting 8.0 and 0.1 μg/kg respectively, from street markets in the Amazon region 6.3 μg/kg and from supermarkets in São Paulo State 0.2 μg/kg. Processing, which included manual or mechanical sorting and drying at 60°C for 30 to 36 h, eliminated on average more than 98% of total aflatoxins. These results showed that sorting is a very effective way to decrease aflatoxin content in brazil nuts.

Molecular identification of Aspergillus spp. isolated from coffee beans

Some species belonging to the genus Aspergillus are potential producers of ochratoxin A (OA), a mycotoxin with nephrotoxic, immunosuppressive, teratogenic and carcinogenic effects. The aim of the present study was to identify the species of Aspergillus that contaminate the inside of coffee beans collected in the stage of maturation and drying, from 16 producing areas located in the northern region of the State of Parana, in the South of Brazil. A total of 108 isolates of Aspergillus spp. was identified at the species level, by sequencing the internal transcribed spacer (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). The results revealed the presence of potentially ochratoxigenic species in 82% of the geographic regions studied, among which Aspergillus niger was the species most frequently detected, followed by A. ochraceus and A. carbonarius. The presence of A. carbonarius in immature coffee fruits harvested from trees is reported for the first time.

Aspergillus bertholletius sp. nov. from Brazil nuts.

During a study on the mycobiota of brazil nuts (Bertholletia excelsa) in Brazil, a new Aspergillus species, A. bertholletius, was found, and is described here. A polyphasic approach was applied using morphological characters, extrolite data as well as partial β-tubulin, calmodulin and ITS sequences to characterize this taxon. A. bertholletius is represented by nineteen isolates from samples of brazil nuts at various stages of production and soil close to Bertholletia excelsa trees. The following extrolites were produced by this species: aflavinin, cyclopiazonic acid, kojic acid, tenuazonic acid and ustilaginoidin C. Phylogenetic analysis using partial β-tubulin and camodulin gene sequences showed that A. bertholletius represents a new phylogenetic clade in Aspergillus section Flavi. The type strain of A. bertholletius is CCT 7615 ( = ITAL 270/06 = IBT 29228).

Brazil nuts are subject to infection with B and G aflatoxin-producing fungus, Aspergillus pseudonomius.

The exploitation of the Brazil nut is one of the most important activities of the extractive communities of the Amazon rainforest. However, its commercialization can be affected by the presence of aflatoxins produced by fungi, namely Aspergillus section Flavi. In the present study, we investigated a collection of Aspergillus nomius strains isolated from Brazil nuts using different approaches, including morphological characters, RAPD and AFLP profiles, partial β-tubulin and calmodulin nucleotide sequences, aflatoxin patterns, as well as tolerance to low water activity in cultured media. Results showed that most of the isolates do belong to A. nomius species, but a few were re-identified as Aspergillus pseudonomius, a very recently described species. The results of the analyses of molecular variance, as well as the high pairwise FST values between A. nomius and A. pseudonomius suggested the isolation between these two species and the inexistence of gene flow. Fixed interspecific nucleotide polymorphisms at β-tubulin and calmodulin loci are presented. All A. pseudonomius strains analyzed produced aflatoxins AFB1, AFB2, AFG1 and AFG2. This study contains the first-ever report on the occurrence in Brazil nuts of A. pseudonomius. The G-type aflatoxins and the mycotoxin tenuazonic acid are reported here for the first time in A. pseudonomius.

Identification of the recently described new type of bovine papillomavirus (BPV-8) in a Brazilian beef cattle herd

Bovine papillomavirus type 8 (BPV-8) was first detected and described in teat warts as well as in healthy teat skin from cattle raised in Japan. The entire viral genome was sequenced in 2007. Additionally, a variant of BPV-8, BPV-8-EB, was also identified from papillomatous lesions of a European bison in Slovakia. In Brazil, despite the relatively common occurrence of BPV infections, the identification and determination of viral types present in cattle is still sporadic. The aim of this study is to report the occurrence of the recently described BPV-8 in Brazil. The virus was identified in a skin warts obtained from a beef cattle herd located in Parana state, southern Brazil. The papilloma had a macular, non-verrucous gross aspect and was located on the dorsal thorax of a cow. Polymerase chain reaction (PCR) was performed using generic primers for partial amplification of L1 gene. The obtained amplicon (480bp) was cloned and two selected clones were sequenced. The nucleotide sequence was compared to existing papillomaviral genomic sequences, identifying the virus as BPV type 8. This study represents the first report of BPV-8 occurrence in Brazil, what suggests its presence among Brazilian cattle.

Multiple bovine papillomavirus infections associated with cutaneous papillomatosis in brazilian cattle herds

Cutaneous papillomatosis is a pathological condition commonly found in cattle and is characterized by the presence of benign proliferative tumors caused by bovine papillomavirus (BPV) infection. While multiple infections with human papillomavirus (HPV) are common in healthy and immunodeficient humans, studies with the aim of identifying mixed infections are still sporadic in veterinary medicine. The aim of this study is to describe the occurrence of multiple BPV infections in cattle affected by cutaneous papillomatosis. Fifteen skin warts were collected from at least two diverse anatomical regions of six bovines with papillomatosis belonging to three cattle herds from the Parana state in Brazil. The BPV types present in the skin wart samples were determined by a PCR assay performed with the FAP primer pair for partial L1 gene amplification followed by direct sequencing or by cloning and sequencing of the inserts. Sequence analysis of the obtained amplicons allowed the identification of four characterized BPV types (BPV-1, -2, -6, and -8) and three previously described putative new BPV types (BPV/BR-UEL3, BPV/BR-UEL4, and BPV/BR-UEL5). Double infections were identified in four (A, B, D, and E) of the six animals included in this study. In this work, the strategy adopted to evaluate skin warts from diverse anatomical sites of the same animal allowed the identification of multiple infections with two or three different BPV types. The analysis of four animals belonging to a single cattle herd also showed the presence of six different viral types. These results clearly suggest that both multiple papillomaviral infection and a high viral diversity can be as frequent in cattle as in human beings.

Genetic relationships among strains of the Aspergillus niger aggregate

We analyzed the genetic relationships between 51 fungal isolates previously identified as A. niger aggregate, obtained from dried fruit samples from worldwide origin and 7 A. tubingensis obtained from Brazilian coffee beans samples. Greater fungal diversity was found in black sultanas. Aspergillus niger sensu stricto was the most prevalent species. It was found in all fruit substrates of all geographical origins. Based on Random Amplification of Polymorphic DNA (RAPD) and β-tubulin sequences data two groups of A. niger were found. In spite of the small number of isolates from Group IV an association between extrolite patterns and molecular clustering is speculated. A. tubingensis were the second most frequent species and this species were clearly subdivided into two groups. The finding of two groups for A. tubingensis strains could not yet explain the contradictions found in the literature about the capability this species for ochratoxin production, because both of them were formed by only non-ochratoxin-producing strains.

Molecular Diagnosis of Ochratoxigenic Fungi

Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins. Its presence in several agricultural commodities has been considered a problem worldwide. This toxin is mainly produced by two genera of fungi: Aspergillus and Penicillium. Ochratoxin A has nephrotoxic, immunosuppressive, and carcinogenic effects; consequently, contamination with OTA presents a major risk for human and animal health. Over the last 5 years, several studies have developed PCR-based assays for identifying and quantifying OTA-producing fungi in food samples. The main objective of these assays is to allow the detection of microorganisms capable of producing OTA, prior to ochratoxin production and accumulation. Several of these attempts will be reviewed and discussed in this chapter.