Marina Venturini Copetti

Ochratoxigenic fungi and ochratoxin A in cocoa during farm processing

This study investigated the occurrence of fungi with the potential to produce ochratoxin A (OTA), and the occurrence of OTA, in Brazilian cocoa beans. Two hundred and twenty two samples of cocoa were evaluated, taken at various stages of fermentation, drying and storage. Samples were collected from Bahia, the main cocoa producing region in Brazil. Fungi with the potential to produce OTA were isolated by direct plating of cocoa beans on Dichloran 18% Glycerol agar after surface disinfection, and identified by standard techniques. The ability of the fungi to produce OTA was estimated using the agar plug technique and TLC. The presence of OTA in cocoa samples was determined by HPLC after immunoaffinity column clean up. The most common ochratoxigenic species found were Aspergillus carbonarius and A. niger aggregate, with lower numbers of A. melleus, A. westerdijkiae and Av. ochraceus. A considerable increase in the numbers of these species was observed during drying and storage. OTA was found at all stages of cocoa processing, with the major incidence during drying and storage. The OTA levels found were in general low and there was a strong positive correlation between the presence of A. carbonarius and OTA contamination in the beans.

Three types of immunotherapics against pythiosis insidiosi developed and evaluated

Pythiosis is a granulomatous disease of horses, cattle, dogs, cats and humans identified in tropical and subtropical areas and caused by Pythium insidiosum, a zoosporic fungus. Experimental models of pythiosis in naturally infected species have not yet been reported but, rabbits may be inoculated with zoospores as an experimental model for studying the disease. The present study evaluates the efficacy of three different of immunotherapics in the rabbit model. Approximately 17500 zoospores of oomycete P. insidiosum (CBS 101555 strain) were inoculated in each animal to generate the disease. Immunotherapics were produced from vortexed or sonicated cultures of the same strain. Four groups of five animals were employed: group 1, placebo; group 2, sonicated immunotherapic; group 3, mixed immunotherapic; and group 4, vortexed immunotherapic. All rabbits were inoculated with viable zoospores one month before administration of the immunotherapics. Eight doses of immunotherapic or placebo were used in each animal with a 14 day interval between injections. Rabbits receiving the vortexed immunotherapic were most effectively protected (P<0.05), showing a decrease in the area of coastal nodules due to Pythiosis insidiosum by 71.8% after 26 weeks of evaluation. Moreover, two animals in this group showed complete remission of the infection at the end of the 26 weeks. In contrast to these findings, rabbits given the sonicated immunotherapic did not show any protection and had an increase of 211.8% in the size of lesions. This failure of sonicated immunotherapic may reflect denaturation of protective antigens due to the sonication method.

Aflatoxigenic fungi and aflatoxin in cocoa

This paper reports the occurrence of aflatoxigenic fungi and the presence of aflatoxins in 226 cocoa samples collected on Brazilian farms. The samples were taken at various stages of fermentation, drying and storage. A total of 819 potentially aflatoxigenic fungi were isolated using Dichloran 18% Glycerol agar after surface disinfection, and identified by standard techniques. The ability of the fungi to produce aflatoxins was determined using the agar plug technique and TLC. The presence of aflatoxins in cocoa samples was determined by HPLC using post-column derivatization with bromide after immunoaffinity column clean up. The aflatoxigenic fungi isolated were Aspergillus flavus, A. parasiticus and A. nomius. A considerable increase in numbers of these species was observed during drying and storage. In spite of the high prevalence of aflatoxigenic fungi, only low levels of aflatoxin were found in the cocoa samples, suggesting the existence of limiting factors to the accumulation of aflatoxins in the beans.

Teste de ELISA indireto para o diagnóstico sorológico de pitiose

Pythiosis is a granulomatous disease caused by the oomycete Pythium insidiosum that affects humans and animals, especially horses. Deaths are very often the consequence of incorrect or late diagnosis when animals no longer respond to treatment. This study aimed standardization of the ELISA assay for the serodiagnostic of pythiosis in horses and rabbits, in order to minimize errors and delays in the diagnosis of the disease. Sera of 72 healthy and 44 of by pythiosis affected horses were used for development and evaluation of the test. The ELISA for equine diagnostic showed 97.72% sensitivity, 90.27% specificity, 86% positive predictive value, 98.4% negative predictive value, and 93.1% efficiency. The rabbit test was standardized with 48 sera of healthy rabbits and 24 sera of rabbits immunized with P. insidiosum antigens. The results were 91.66 % sensitivity, 95.83% specificity, 91.66% positive predictive value, 95.83% negative predictive value, and 94.44% efficiency. It can be concluded that ELISA is a reliable test for diagnostic and serological monitoring of pythiosis.

Occurrence of ochratoxin A in cocoa by-products and determination of its reduction during chocolate manufacture

This work reports an investigation carried out to assess the natural occurrence of ochratoxin A in 168 samples from different fractions obtained during the technological processing of cocoa (shell, nibs, liquor, butter, cake and cocoa powder) and the reduction of ochratoxin A during chocolate manufacture. Ochratoxin A analyses were performed with immunoaffinity columns and detection by high performance liquid chromatography. Concerning the natural ochratoxin A contamination in cocoa by-products, the highest levels of ochratoxin A were found in the shell, cocoa powder and cocoa cake. The cocoa butter was the least contaminated, showing that ochratoxin A seems to remain in the defatted cocoa solids. Under the technological conditions applied during the manufacture of chocolate in this study and the level of contamination present in the cocoa beans, this experiment demonstrated that 93.6% of ochratoxin A present in the beans was reduced during the chocolate producing.

Fungi and mycotoxins in cocoa: from farm to chocolate.

Cocoa is an important crop, as it is the raw material from which chocolate is manufactured. It is grown mainly in West Africa although significant quantities also come from Asia and Central and South America. Primary processing is carried out on the farm, and the flavour of chocolate starts to develop at that time. Freshly harvested pods are opened, the beans, piled in heaps or wooden boxes, are fermented naturally by yeasts and bacteria, then dried in the sun on wooden platforms or sometimes on cement or on the ground, where a gradual reduction in moisture content inhibits microbial growth. Beans are then bagged and marketed. In processing plants, the dried fermented beans are roasted, shelled and ground, then two distinct processes are used, to produce powdered cocoa or chocolate. Filamentous fungi may contaminate many stages in cocoa processing, and poor practices may have a strong influence on the quality of the beans. Apart from causing spoilage, filamentous fungi may also produce aflatoxins and ochratoxin A. This review deals with the growth of fungal species and formation of mycotoxins during the various steps in cocoa processing, as well as reduction of these contaminants by good processing practices. Methodologies for fungal and mycotoxin detection and quantification are discussed while current data about dietary exposure and regulation are also presented.

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).

The effect of cocoa fermentation and weak organic acids on growth and ochratoxin A production by Aspergillus species

The acidic characteristics of cocoa beans have influence on flavor development in chocolate. Cocoa cotyledons are not naturally acidic, the acidity comes from organic acids produced by the fermentative microorganisms which grow during the processing of cocoa. Different concentrations of these metabolites can be produced according to the fermentation practices adopted in the farms, which could affect the growth and ochratoxin A production by fungi. This work presents two independent experiments carried out to investigate the effect of some fermentation practices on ochratoxin A production by Aspergillus carbonarius in cocoa, and the effect of weak organic acids such as acetic, lactic and citric at different pH values on growth and ochratoxin A production by A. carbonarius and Aspergillus niger in culture media. A statistical difference (ρ<0.05) in the ochratoxin A level in the cured cocoa beans was observed in some fermentation practices adopted. The laboratorial studies demonstrate the influence of organic acids on fungal growth and ochratoxin A production, with differences according to the media pH and the organic acid present. Acetic acid was the most inhibitory acid against A. carbonarius and A. niger. From the point of view of food safety, considering the amount of ochratoxin A produced, fermentation practices should be conducted towards the enhancement of acetic acid, although lactic and citric acids also have an important role in lowering the pH to improve the toxicity of acetic acid.

Pulmonary aspergillosis outbreak in Rhea americana in Southern Brazil

Commercial raising of rheas is currently in expansion in the south of Brazil,and many diseases previously restricted to other avian species are currentlyemerging on rhea farms, especially as a result of careless management of theseanimals. The objective of the present article is to report a pulmonary aspergillosisoutbreak that occurred in great rhea (Rhea americana) in the south of Brazil.About 50 birds aged 30 to 60 days died suddenly and one of them was submittedto autopsy which revealed the presence of white caseous nodules 0.5 mm in diameter occupying 95% of the lung area. One lung was sent to the FederalUniversity of Santa Maria for histopathological and mycological analyses.Histopathological analysis revealed multifocal areas with necrosis and inflammatoryinfiltrates and the presence of fungal hyphae, giant cells and fibrous tissue proliferationat the periphery. Aspergillus fumigatus was recovered as pure culture from all culture media. This appears to be the first report of aspergillosis among great rheain Brazil and the second in the world.

Pneumonia intersticial em bovinos associada à ingestão de batata-doce (Ipomoea batatas) mofada

Cases of respiratory disease were diagnosed in five out of 23 cattle (21.7%) after they were fed moldy damaged sweet potatoes (Ipomoea batatas) on a small farm in the county of Sao Vicente do Sul, state of Rio Grande do Sul, Brazil. Of those five cattle, three died spontaneously and another one was euthanatized for necropsy while showing advanced respiratory clinical signs. The disease manifested itself approximately 24 hours after the ingestion of the sweet potatoes and lasted from 1 to 4 days. Clinical signs included dyspnea (labored breathing and abdominal respiration), tachypnea, extended neck with low carriage of the head and rhythmical flaring of the nostrils. Two cows were necropsied. Necropsy findings included distended pale and rubbery lungs which failed to collapse when the thorax was open, and marked pulmonary interstitial emphysema and edema. Lymphoid hyperplasia was observed in the hilar nodes and spleen. Histologically, the lesions were those of interstitial pneumonia. Alveolar septa were thickened by fibroblasts and inflammatory cells, and there was hypertrophy and hyperplasia of type II pneumocytes; the interlobular septa were distended by edema and emphysema. The culture of the moldy sweet potatoes yielded Fusarium solani and Fusarium oxysporum.