Abstract A Penicillium sp. strain was isolated from corroded parts in the Coaracy Nunes hydroelectric power plant in Amapá State, Brazil. Morphological and molecular techniques identified this strain as Penicillium citreosulfuratum Biourge, whose 5.8S-ITS rDNA sequence grouped well in a phylogenetic tree with other Exilicaulis section species of the Penicillium genus. The obtained strain of P. citreosulfuratum ability to form biofilm on the surface of aluminum wires was confirmed by optical and scanning electron microscopy. In plates containing PDA or T&K media, the P. citreosulfuratum strain growth was inhibited around a paper disk containing a highly concentrated solution of copper, iron, or nickel. However, there was no inhibition halo around the paper disk containing aluminum and there was a faint halo around the paper disk containing zinc. This fungus was able to grow in a modified T&K liquid medium containing 50 mM of Al3+ and less efficiently in the same medium but containing 50 mM Zn2+. The essential oils of melaleuca, mint, thyme, and oregano at 100% inhibited the fungus growth. Oregano essential oil inhibited the P. citreosulfuratum strain growth in concentrations as low as 10%. In conclusion, the results show that the obtained strain of P. citreosulfurarum can form a biofilm on aluminum wires, it grows in the presence of a high concentrations of aluminum and zinc, and essential oils can control its growth. sp State Brazil Biourge 5.8SITS 58SITS SITS 5.8S ITS 5 8S S genus P microscopy TK T K media copper iron nickel However zinc Al3 Al Zn2 Zn Zn2+ melaleuca mint thyme 100 10 10% conclusion 8SITS 58S 1

ABSTRACT The presence of mycotoxins or related fungi in animal feed is a major problem for animal and human health. Silage and concentrated feed samples were collected from 21 dairy farms in the Western part of Paraná state in Southern Brazil. Water activity and pH of all samples were measured, and each sample was analyzed to check for the presence of aflatoxigenic Aspergillus. Water activity was observed to be lower in the concentrated feed samples. The pH was lower in the silage samples, indicating fermentation processes. Two silage samples and four concentrated feed samples were contaminated with Aspergillus spp. Seven isolates of Aspergillus spp. were obtained and their potential to produce aflatoxins was evaluated. Four of the isolates, two from the silage samples and two from the concentrated feed samples, produced the aflatoxins B1, B2, G1, and G2 in culture media. These isolates were identified as Aspergillus parasiticus and Aspergillus nomius. The presence of aflatoxigenic isolates of Aspergillus spp. in silage and concentrated feed samples is a matter of concern, because of the risk of aflatoxin production and contamination of the animal feed.

Abstract Aflatoxins are mutagenic, carcinogenic, and teratogenic mycotoxins. The objective of this work was to study the presence of aflatoxigenic Aspergillus in commercial Bulgur wheat in the city of Maringá, Paraná, Brazil. Thirty samples of commercial Bulgur wheat, acquired in the period of August 2011 to January 2012, were evaluated. The enumeration analysis showed that samples had up to 273.3 CFU of molds and 133.3 CFU of aflatoxigenic Aspergillus per gram of wheat. Forty-two monosporic isolates were obtained and identified as Aspergillus flavus. The isolates were analyzed regarding their aflatoxigenic potential by culture in coconut milk agar; hydroxide vapor exposure; chromatography; and polymerase chain reaction (PCR) targeting genes that code enzymes of the aflatoxins synthesis pathway. Some of the isolates were confirmed to be aflatoxin producers and several of them presented a genetic profile of aflatoxin synthesis. The obtained results demonstrated that Bulgur wheat A. flavus contamination is concerning.

The productivity of wheat and corn crops depends on climatic conditions and resistance against phytopathogenic fungi such as those of the genus Fusarium. Some species of this genus produce zearalenone (ZEA), a mycotoxin with hyperestrogenic effects. The objective of this study was to investigate the presence of ZEA in samples of cracked wheat (n = 109), popcorn (n = 51) and corn grits (n = 50) commercialized in the State of Paraná, Brazil. Commercial samples of each crop were collected between September 2007 and June 2008 and analyzed by thin-layer chromatography. The method used for detection of the mycotoxin in wheat and corn derivatives presented a recovery rate of 94.5% and 99.5%, respectively, detection limit of 40 μg.kg-1 and quantification limit of 55 μg.kg-1. No contamination with ZEA was detected in cracked wheat samples. Among the corn derivatives, only one cracked corn sample was contaminated with ZEA (64 μg.kg-1). Despite the low contamination observed, monitoring the occurrence of mycotoxins in foods is important to ensure safety.

A detecção de fungos micotoxigênicos em alimentos é importante porque sua presença pode indicar uma possível contaminação com as micotoxinas associadas. Fusarium graminearum é um patógeno de trigo e um produtor de micotoxinas. A reação da polimerase em cadeia (PCR) é empregada na identificação específica de F. graminearum. No entanto, essa metodologia não tem sido comumente empregada na detecção de F. graminearum em alimentos. Assim, o presente trabalho teve como objetivo desenvolver uma metodologia molecular para detectar F. graminearum em amostras comerciais de trigo para quibe. Dois métodos foram testados. No primeiro, uma amostra desse cereal foi contaminada com micélio de F. graminearum. O DNA genômico foi extraído dessa mistura e utilizado em uma reação de PCR específica para F. graminearum. A espécie F. graminearum foi detectada somente em amostras altamente contaminadas. No segundo método, amostras de trigo para quibe foram inoculadas em um meio de cultura sólido e isolados com características culturais de F. graminearum foram obtidos. O DNA extraído desses isolados foi utilizado em reações de PCR específicas para F. graminearum. Um isolado obtido teve o genótipo de produção de tricotecenos determinado por PCR. A metodologia estabelecida poderia ser utilizada em levantamentos de contaminação de alimentos com F. graminearum.

The detection of mycotoxigenic fungi in foodstuff is important because their presence may indicate the possible associated mycotoxin contamination. Fusarium graminearum is a wheat pathogen and a producer of micotoxins. The polymerase chain reaction (PCR) has been employed for the specific identification of F. graminearum. However, this methodology has not been commonly used for detection of F. graminearum in food. Thus, the objective of the present study was to develop a molecular methodology to detect F. graminearum in commercial samples of bulgur wheat. Two methods were tested. In the first method, a sample of this cereal was contaminated with F. graminearum mycelia. The genomic DNA was extracted from this mixture and used in a F. graminearum specific PCR reaction. The F. graminearum species was detected only in samples that were heavily contaminated. In the second method, samples of bulgur wheat were inoculated on a solid medium, and isolates having F. graminearum culture characteristics were obtained. The DNA extracted from these isolates was tested in F. graminearum specific PCR reactions. An isolate obtained had its trichothecene genotype identified by PCR. The established methodology could be used in surveys of food contamination with F. graminearum.

O principal objetivo deste trabalho foi desenvolver um novo protocolo de PCR para identificação de isolados de Fusarium graminearum, baseado no uso de um par de iniciadores direcionado para um segmento da região 3' codificadora do gene gaoA que codifica a enzima galactose oxidase (GO). Esta região possui baixa homologia com a mesma região de genes da GO de outros fungos. O DNA genômico de 17 cepas de Fusarium spp. isoladas de cereais infectados com sintomas, de vários outras espécies de Fusarium e de outros gêneros de fungos foi analisado em um protocolo de PCR utilizando os iniciadores desenhados. Os 17 isolados de Fusarium spp. também foram analisados para a produção da enzima GO em fermentação submersa em um novo meio líquido. Todas as cepas que foram morfologicamente e molecularmente identificadas como F. graminearum foram capazes de secretar a enzima e tiveram um resultado positivo no protocolo de PCR, utilizando os iniciadores direcionados para o gene gaoA. Nenhum fragmento de DNA foi amplificado quando foi utilizado o DNA genômico de várias outras espécies de Fusarium e de espécies de outros gêneros de fungos. Os resultados sugerem que o protocolo de PCR gerado é específico e pode ser considerado como uma nova ferramenta molecular para a identificação de cepas de F. graminearum. Além disso, o meio líquido formulado é uma alternativa barata para a avaliação da produção de GO por F. graminearum.

The main objective of this work was to develop a PCR protocol for the identification of Fusarium graminearum, based on a pair of primers targeted to a segment of the 3' coding region of the gaoA gene that codes for the enzyme galactose oxidase (GO). This region has low homology with the same region of GO genes from other fungi. Genomic DNA from 17 strains of Fusarium spp. isolated from diseased cereals, from several other Fusarium species, and from other fungi genera was analyzed in a PCR assay using this primer set. The 17 strains of Fusarium spp. were also analyzed for the GO enzyme production in submerse fermentation in a new formulated liquid medium. All strains that were morphologically and molecularly identified as F. graminearum were able to secrete the enzyme and had a positive result in the used PCR protocol. No DNA fragment was amplified using genomic DNA from other Fusarium species and species of other fungi genera. The results suggest that the proposed PCR protocol is specific and can be considered as a new molecular tool for the identification of F. graminearum. In addition, the new formulated medium is a cheap alternative for screening for GO screening production by F. graminearum.

Uma análise visando encontrar novos isolados produtores de galactose oxidase (GO) e avaliar a distribuição da produção entre cepas de Fusarium graminearum foi conduzida. Trinta e cinco isolados de 39 testados produziram a enzima em diferentes níveis. Os dados indicaram uma ampla distribuição da produção da galactose oxidase por F. graminearum e revelaram novos isolados produtores. A enzima produzida por diferentes isolados apresentou atividade e estabilidade térmicas similares e foi ativa sobre os mesmos substratos. No entanto, o pH ótimo variou de 7,0 a 7,5. Assim, todos os isolados são adequados para a produção de galactose oxidase.

A screening aimed to find new galactose oxidase producer isolates and to evaluate the production among Fusarium graminearum strains was conducted. Thirty-five isolates out of 39 analysed produced the enzyme at several levels. The data indicated a wide distribution of galactose oxidase within F. graminearum and also revealed new producer isolates. The enzyme produced by different isolates showed similar thermal activity and stability and were active on same substrates. However, the optimum pH ranged from 7.0 to 7.5. Thus, all evaluated isolates were suitable for the production of galactose oxidase.

The objective of this work was to compare the polymerase chain reaction (PCR) using lesion scrapping with other conventional techniques for the diagnosis of the American tegumentary leishmaniasis (ATL). For this, patients with cutaneous lesions suspected to be ATL were studied. The DNA was amplified with the MP1L/MP3H primers. From the 156 studied patients, 79 (50.6%) presented positive parasite direct search (PD), 81 (51.9%) had positive Montenegro skin test (MST), and 90 (57.7%) presented PD and/or MST positive. The PCR was positive in all of the positive-PD patients (100% sensitivity), in 91.1% of the positive PD and/or MST patients, and in 27.3% of the patients that presented negative PD and positive MST. The PCR positivity was similar to the PD (P = 0.2482) and inferior to the MST (P = 0.0455), and to the PD/MST association (P = 0.0133). The high PCR sensitivity, and positivity in those cases where the PD was negative, highlights the importance of this technique as an auxiliary tool for the diagnosis of ATL.

A leishmaniose tegumentar americana (LTA) foi estudada em 143 cães da área rural no Município de Mariluz, noroeste do Estado do Paraná, Brasil, utilizando a pesquisa direta do parasito (PD), a imunofluorescência indireta (IFI) e a reação em cadeia da polimerase (PCR). Trinta e nove cães (27,3%) apresentavam lesões sugestivas da doença, 5 (12,8%) deles foram positivos na PD e PCR em tecido de lesão, e quatro foram também positivos na IFI. Dos 34 cães com PD negativa, 12 (35,3%) tiveram a PCR (lesão) positiva, e cinco desses tiveram também IFI positiva. Cento e quatro cães não apresentavam lesão, mas 17/101 (16,8%) tiveram IFI positiva. A PCR no sangue foi positiva em 10/38 (26,3%) cães com lesão e em 16/104 (15,4%) sem lesão. A associação entre PCR (lesão ou sangue), PD e IFI detectou 24/39 (61,5%) positivos entre os cães sintomáticos e 31/104 (29,8%) positivos entre os assintomáticos. A PCR foi útil para o diagnóstico de LTA, não houve relação entre presença de lesão, sorologia e PCR no sangue, e a detecção de DNA do parasito no sangue pode indicar a ocorrência de disseminação hematogênica do parasito.

American tegumentary leishmaniasis (ATL) was studied in 143 dogs in a rural area in the county of Mariluz, northwestern Paraná State, Brazil, using direct parasite search, indirect immunofluorescence (IIF), and polymerase chain reaction (PCR). Thirty-nine dogs (27.3%) presented lesions suggestive of the disease, 5 (12.8%) of which were positive in direct parasite search and PCR (lesion), and of these 5, 4 were also positive by IIF. Of the 34 dogs with negative direct parasite search, 12 (35.3%) had PCR- positive lesions, and of these, 5 were also IIF-positive. One hundred and four dogs had no lesions, but 17/101 (16.8%) were IIF-positive. PCR in blood was positive in 10/38 (26.3%) of the dogs with lesions and in 16/104 (15.4%) of dogs without lesions. The association between PCR (lesion or blood), direct parasite search, and IIF detected 24/39 (61.5%) positive results among symptomatic dogs and 31/104 (29.8%) among asymptomatic animals. PCR was useful for diagnosing ATL, but there was no correlation between lesions, serology, and plasma PCR. Furthermore, detection of parasite DNA in the blood may indicate hematogenous parasite dissemination.

American tegumentary leishmaniasis (ATL) was studied in 143 dogs in a rural area in the county of Mariluz, northwestern Paraná State, Brazil, using direct parasite search, indirect immunofluorescence (IIF), and polymerase chain reaction (PCR). Thirty-nine dogs (27.3%) presented lesions suggestive of the disease, 5 (12.8%) of which were positive in direct parasite search and PCR (lesion), and of these 5, 4 were also positive by IIF. Of the 34 dogs with negative direct parasite search, 12 (35.3%) had PCR- positive lesions, and of these, 5 were also IIF-positive. One hundred and four dogs had no lesions, but 17/101 (16.8%) were IIF-positive. PCR in blood was positive in 10/38 (26.3%) of the dogs with lesions and in 16/104 (15.4%) of dogs without lesions. The association between PCR (lesion or blood), direct parasite search, and IIF detected 24/39 (61.5%) positive results among symptomatic dogs and 31/104 (29.8%) among asymptomatic animals. PCR was useful for diagnosing ATL, but there was no correlation between lesions, serology, and plasma PCR. Furthermore, detection of parasite DNA in the blood may indicate hematogenous parasite dissemination.

A leishmaniose tegumentar americana (LTA) foi estudada em 143 cães da área rural no Município de Mariluz, noroeste do Estado do Paraná, Brasil, utilizando a pesquisa direta do parasito (PD), a imunofluorescência indireta (IFI) e a reação em cadeia da polimerase (PCR). Trinta e nove cães (27,3%) apresentavam lesões sugestivas da doença, 5 (12,8%) deles foram positivos na PD e PCR em tecido de lesão, e quatro foram também positivos na IFI. Dos 34 cães com PD negativa, 12 (35,3%) tiveram a PCR (lesão) positiva, e cinco desses tiveram também IFI positiva. Cento e quatro cães não apresentavam lesão, mas 17/101 (16,8%) tiveram IFI positiva. A PCR no sangue foi positiva em 10/38 (26,3%) cães com lesão e em 16/104 (15,4%) sem lesão. A associação entre PCR (lesão ou sangue), PD e IFI detectou 24/39 (61,5%) positivos entre os cães sintomáticos e 31/104 (29,8%) positivos entre os assintomáticos. A PCR foi útil para o diagnóstico de LTA, não houve relação entre presença de lesão, sorologia e PCR no sangue, e a detecção de DNA do parasito no sangue pode indicar a ocorrência de disseminação hematogênica do parasito.