Natural occurrence of Fusarium species and fumonisin-production by toxigenic strains isolated from poultry feeds in Argentina

Fusarium species and fumonisin production by toxigenic strains were investigated. During 1996–1998, 158 samples of poultry feeds were collected from a factory located in the department of Río Cuarto Córdoba province, Argentina. The most common species of Fusarium were F. moniliforme (60.7%) and F. nygamai (35.4%) followed by F. semitectum, F. subglutinans, F. proliferatum, F. dlamini, F. solani, F. oxysporum and F. napiforme. Fungal counts ranged from 1 × 10³ to 8 × 10⁵ CFU/g with mean values from 1.5 × 10³ to 2.3 × 10⁵ CFU/g. The highest counts were for F. dlamini, F. subglutinans, F. moniliforme and F. nygamai. Strains of F. moniliforme, F. nygamai, and F. proliferatum were screened for their potential to produce fumonisin B₁ (FB₁), fumonisin B₂ (FB₂) and fumonisin B₃ (FB₃) in corn grain. The samples were analysed using a modified high performance liquid chromatography method. The strains assayed, 43 strains, produced three fumonisins. There was a high degree of variability in the quantities of FB₁, FB₂, and FB₃ produced. The toxin produced in highest levels by the majority of the strains was FB₁. The range of concentration varied from 5.4 to 3,991, 1.01 to 189 and 0.4 to 765 ppm per gram of corn for FB₁, FB₂ and FB₃ respectively. The toxigenic pattern of strains was normal, although two strains of F. moniliforme produced exceptionally high concentrations of FB₃ and minor concentrations of FB₂ and FB₁. This is the first report from Argentina on Fusarium species in poultry feeds and fumonisin production by these strains.This revised version was published online in October 2005 with corrections to the Cover Date.     

Aflatoxin-producing potential of Aspergillus flavus strains isolated from Spanish poultry feeds

A total of 126 fungal strains belonging to the Aspergillus flavus group isolated from commercial poultry mixed feeds were studied. One hundred and twenty-five were identified as A. flavus and one as A. parasiticus. Forty nine strains (39%) produced aflatoxins on a crushed moist wheat medium (28 °C/10 days), whereas only sixteen (13%) showed specific fluorescence on Aflatoxin-Producing Ability Medium. In both media, mainly aflatoxins B₁ and B₂ were detected, the average concentration of aflatoxins being 4294+/–1083 g/kg in crushed moist wheat medium, and 877+/–257 g/kg in Aflatoxin-Producing Ability Medium.     

Mycoflora and naturally occurring mycotoxins in poultry feeds in Argentina

The purpose of this work was to determine the mycoflora and mycotoxins natural incidence in poultry feeds from 2 factories in Río Cuarto, Córdoba. One hundred and thirty samples were taken from May/1996 to May/1997. The most dominant species isolated of poultry feed samples belonged to the genera Aspergillus spp 85% and Fusarium spp 70%. From Aspergillus genus eleven species were identified and A. flavus was the most frequent. Nine species were identified from the Fusarium genus and the predominant was F. moniliforme. Penicillium ranked third in the number of isolated cases. From this genus twelve species were collected of which P. brevicompactum (15%), P. restrictum (14%) and P. purpurogenum (12%) were the most common.The most significant mycotoxin from poultry feeds was aflatoxin B₁ (AFB₁) found in 48% of the samples, with levels ranging from 10 to 123 ng/g. For zearalenone (ZEA) the levels were 327 to 5,850 ng/g and DON was not detected from the samples. Due to the fact that in Argentina there is little information about this topic, these data on poultry feeds in our region would be of worldwide interest.This revised version was published online in October 2005 with corrections to the Cover Date.     

A mycological and bacteriological survey on feed ingredients and mixed poultry feeds in Reunion island

A survey was carried out in Reunion island to obtain data on the occurrence of fungi, aflatoxigenic strains of Aspergillus flavus, aflatoxins, total aerobic bacteria and salmonellae of 150 samples of mixed poultry feeds and raw materials. These were collected at five farms over a 3-month period during the warm rainy season.White corn and Brazilian soybean meal seemed to present a better microbiological quality than yellow corn and US soybean meal.Mixed poultry feeds presented a high total mold count reflecting the mold flora of raw materials. The most frequent and abundant fungi were Aspergillus flavus, A. glaucus group, Fusarium spp., Penicillium spp., A. candidus, Mucor spp., A. restrictus, Scopulariopsis spp., Cladosporium spp. and A. versicolor. Of the 118 A. flavus strains screened, 42 (35.6%) were aflatoxigenic. Yellow corn samples were the most frequently contamined with aflatoxigenic strains (54.5%), followed by mixed feeds (44%).Of the 66 samples tested, 24 (36%) contained aflatoxins (traces to 22 ng/g). A good correlation seemed to exist between presence of at least one aflatoxigenic strain per sample and presence of aflatoxins.     

Minimal moisture content for growth and aflatoxin production by Aspergillus parasiticus in mixed feeds

Minimal moisture content for growth and aflatoxin production by Aspergillus parasiticus in mixed feeds was studied. Minimal moisture content for growth is 16.51%+/–0.45. Very low amounts of aflatoxins are accumulated at days 1 or 2 after the growth started when the initial moisture content of the mixed feed was 17% or lower; on the other hand, significant amounts of aflatoxin are detected when it was 18% or higher.     

Influence of carbon and nitrogen sources on ochratoxin A production by two species of Penicillium isolated from poultry feeds

Mycotoxins are natural, secondary metabolites produced by fungi on agricultural commodities in the field and during storage under a wide range of climatic conditions. The ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by several species of Aspergillus and Penicillium. In this study, the influence of carbon and nitrogen sources on ochratoxigenic Penicillium species was assessed. The ochratoxigenic Penicillium species were isolated from poultry feed samples of Andhra Pradesh, India. The isolated ochratoxigenic Penicillium species were identified, screened and characterised as OTA producers by high performance thin layer chromatography (HPTLC) and confirmed by high performance liquid chromatography (HPLC). This experiment was carried out using Czapak yeast Autolysate (CYA) medium with different carbon (C) and nitrogen (N) sources at pH 6.5 and incubated at 25 ± 2°C under dark condition. Maximum OTA production was recorded in the presence of D-glucose followed by D-galactose and D-lactose as carbon sources. Similarly, the maximum amount of OTA production was observed in thiourea followed by potassium nitrate as nitrogen source. However, OTA production, final pH of the medium, and mycelial yield and OTA production of both the species of Penicillium varied with C and N present in the medium. The kinetics of the both species of Penicillium was observed for 30 days at an interval of three days. The maximum amount of OTA was detected by 12 and 15 days incubation periods for Penicillium nordicum and Penicillium verrucosum, respectively.     

Considerations on the distribution of aflatoxigenic Aspergillus flavus in feeds

The distribution of aflatoxin producing isolates of the Aspergillus flavus group in feeds was studied. Aflatoxin production was investigated by a sequential method previously reported (fluorescence in Coconut Agar Medium, rapid extraction from a wheat medium, and total extraction from the same wheat medium). Twenty-seven of 32 samples contained A. flavus, and 21 of them had at least one aflatoxicogenic isolate of A. flavus. Of the 115 isolates analysed, 65 produced aflatoxins, mainly B aflatoxins.     

Mycoflora and incidence of aflatoxin B1, zearalenone and deoxynivalenol in poultry feeds in Argentina

In Argentina, there is rather little information about the natural occurrence of mycotoxins in feedstuffs. The aim of this work was to determine the fungal flora and natural incidence of aflatoxin B1 (AFB1), zearalenone (ZEA) and deoxynivalenol (DON) in poultry feeds from 5 factories of Río Cuarto, Córdoba. Three hundred samples were taken from May 1995 to May 1996. Fungal counts of poultry feeds ranged 104 to 106 CFU g-1. The lowest counts were obtained on the first months from the sampling (May to September 1995) with mean values significantly different from those found at the last of the sampling (October 1995 to April 1996). The most prevalent species isolated of poultry feed samples belonged to the genera Penicillium that was present in 98% of the samples, Fusarium (87%) and Aspergillus (52%). Fusarium species isolated were: F moniliforme in 73% of the samples, F subglutinans (35%), F graminearum (20%) and within Aspergillus species: A. parasiticus (33%) and A. flavus (8%) were identified. In poultry feeds aflatoxin B1 (AFB1) was the most significant mycotoxin with levels ranging from 17 to 197 ng/g. For deoxynivalenol (DON) the levels ranged from 240 to 410 ng/g. Only three out of 300 samples were contaminated with zearalenone (ZEA) in concentrations of 30, 120 and 280 ng/g. These are preliminary data on this subject in our region. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic diversity in Aspergillus parasiticus population from the peanut agroecosystem in Argentina

AIMS: The aims of this work were to identify potential sources of Aspergillus parasiticus inoculum and to evaluate the sclerotial and toxigenic profiles of this species from the peanut agroecosystem in Argentina. Likewise, the genetic diversity of A. parasiticus population was analysed using vegetative compatibility group (VCG) analysis. METHODS AND RESULTS: The A. parasiticus strains were isolated from soil, debris and peanut seeds in Córdoba Province, Argentina. A. parasiticus was recovered from the three sources analysed. Only 11 of 185 A. parasiticus isolates (5.9%) did not produce aflatoxins, while 57% produced sclerotia. Twenty-four VCG were identified from 63 isolates. The VCG diversity index for A. parasiticus, expressed as the number of groups divided by the total number of isolates, was 0.31. In general, there were significant differences among VCG in aflatoxin production. CONCLUSIONS: The presence of aflatoxigenic strains of A. parasiticus in the three substrates suggests that they may be an important source of aflatoxin in Argentina's peanut agroecosystem. The A. parasiticus population shows a low genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study showed data on inoculum distribution, aflatoxin and sclerotia production and genetic diversity in an A. parasiticus population isolated from the peanut agroecosystem in Argentina.     

Cyclopiazonic acid production by submerged cultures of Penicillium and Aspergillus strains

Abstract 62 isolates of Penicillium and Aspergillus were screened for cyclopiazonic acid (CPA) production by surface and submerged culture on different media. The production of this mycotoxin was restricted to Penicillium camembertii group II (and its domesticated form P. camembertii ), P. griseofulvum , and Aspergillus flavus (and its domesticated form A. oryzae ). The best yield of CPA was obtained by a strain of P. griseofulvum , but several strains of P. camembertii group II were also good producers. Propionic acid (500 and 1000 mg/l medium) did not enhance the production of CPA. The best yields of CPA were obtained in submerged culture, but in some cases growth and CPA production only occured in surface culture. A simplified procedure for isolation of CPA is described.     

Molecular characterization of Aspergillus section Flavi isolates collected from peanut fields in Argentina using AFLPs

AIMS: The objectives of this study were: (i) to evaluate genetic relatedness among Aspergillus section Flavi strains isolated from soil and peanut seeds in Argentina; (ii) to determine if AFLP molecular markers could be useful to identify isolates up to species level, and to correlate these markers with the isolates' toxigenic potentials and/or vegetative compatibility group (VCG) affiliations. METHODS AND RESULTS: Amplified fragment length polymorphism (AFLPs) analysis was applied to compare 82 isolates of Aspergillus section Flavi. Cluster analysis showed a clear separation of A. flavus and A. parasiticus, and comparison of fingerprints revealed several specific markers for each group of isolates. AFLP analysis indicates that no genotypical differences can be established between aflatoxigenic and nonaflatoxigenic producers in both species analysed. In addition, candidate AFLP markers associated with a particular VCG were not found. CONCLUSIONS: There was a concordance between morphological identification and separation up to species level using molecular markers. The findings of specific bands for A. flavus and A. parasiticus may be useful for the design of specific PCR primers in order to differentiate these species and detect them in food. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides new data on molecular characterization of Aspergillus section Flavi in Argentina.     

Occurrence of fumonisin B1 in maize and poultry feeds in Haryana,India

A total of 100 maize and 50 poultry feed samples collected in 1998 at random from nine and eight districts of Haryana, respectively, were analysed for fumonisin B₁. The samples were collected from poultry farms, feed manufacturers and markets. Ninety one (91%) maize samples and forty two (84%) poultry feed samples were found to contain fumonisin B₁. Fumonisin B₁ contamination in the maize samples ranged from 0.1–87.0 ppm. Whereas the poultry feed samples contained fumonisin B₁ in the range of 0.02–28.0 ppm. It indicated widespread prevalence of fumonisin B₁ in maize and poultry feeds in different areas of Haryana. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of some screening methods for aflatoxigenic moulds

Thirty seven strains of the Aspergillus flavus group isolated from animal mixed feeds have been screened for their ability to produce aflatoxins in yeast extract and sucrose (YES), aflatoxin producing ability (APA), and coconut agar medium (CAM) media. The concentration and detection of the aflatoxins by different methods is compared. Five known aflatoxin-positive and one aflatoxin-negative strains have been used as controls. Only 5 out of the 37 strains (13.5%) were aflatoxin-producers in YES medium. Of these five strains and the five known aflatoxin-positive strains, only three showed blue fluorescence in APA medium and four in CAM medium. Generally, the aflatoxin concentration in CAM medium was higher than in YES and APA media. Using the agar-plug method and by direct spotting of the YES broth on TLC plates, some aflatoxin-producing strains were not detected.     

Mycoflora and ochratoxin-producing strains of Aspergillus section Nigri in wine grapes in Argentina

AIMS: The aims of this work were to evaluate the mycoflora and to identify the species of Aspergillus with the potential to produce ochratoxin A (OA) from different wine grape varieties from Mendoza, Argentina. Likewise, the capacity to produce OA by Aspergillus section Nigri was studied. METHODS AND RESULTS: Fifty samples of wine grapes were obtained from a winery of Mendoza province, Argentina. The surface-disinfection method was used for mycoflora determination using the medium dichloran 18% glycerol agar (DG18). Alternaria, Aspergillus and Penicillium were identified at species level. OA production was tested in 63 strains belonging to section Nigri. Alternaria genus was the most frequent (80% of the samples) followed by Aspergillus (70%). Alternaria alternata was the only specie identified from the Alternaria genus, followed by A. niger var. niger, A. flavus among others. From Penicillium genus, P. crysogenum was the most frequent specie. From 63 strains of Aspergillus section Nigri, 41.3% were OA producers. The levels of produced toxin ranged from 2 to 24.5 ng ml-1 of culture medium. CONCLUSIONS: The presence of ochratoxigenic strains of Nigri section in this substrate suggests that they may be an important source of OA in grapes from tropical and subtropical zones. Therefore, the industry should work further to diminish the growth of these fungi and mycotoxins formation in grapes, with the aim to reduce OA content in wine products. SIGNIFICANCE AND IMPACT OF THE STUDY: The wine grape contamination with A. alternata and Aspergillus section Nigri was significant.     

Enumeration of thermotolerant Campylobacter spp. from poultry carcasses at the end of the slaughter-line

AIM: To enumerate Campylobacter on poultry carcasses at the end of the slaughter-line, and investigate the extent to which Campylobacter from a positive flock were transmitted to other flocks during slaughter. METHODS AND RESULTS: The presence (in caeca) and the level (from carcasses) of Campylobacter were determined. The isolates were fingerprinted by amplified fragment length polymorphism (AFLP). A total of three of 13 broiler flocks and three of four-layer flocks harboured caecal Campylobacter. Carcasses from the caeca-positive broiler flocks were Campylobacter positive with numbers ranging from 2.6 x 10(4) to 2.6 x 10(6) CFU per carcass. Two caeca-negative broiler flocks, slaughtered directly after the positive broiler flocks, had the first carcasses contaminated with Campylobacter, with numbers below 2 x 10(4) CFU per carcass of the same AFLP haplotypes as the preceding flock. Campylobacter was detected on carcasses from only one of the caeca-positive layer flocks in numbers below 2 x 10(4) CFU per carcass. No Campylobacter was detected on carcasses from a flock succeeding the positive-layer flocks. CONCLUSION: Carcasses from Campylobacter-positive broiler flocks were heavily contaminated with Campylobacter, and transmitted low levels of Campylobacter to carcasses from negative flocks, slaughtered directly after. Campylobacter-positive layer flocks had low numbers of Campylobacter on the carcasses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate limited cross-contamination of Campylobacter between flocks at the slaughterhouse, reducing the advantage of logistic slaughter.     

Potency of plant extracts against Penicillium species isolated from different seeds and fruits in Saudi Arabia

Antifungal activity of extracts of cinnamon (Cinnamomum zeylanicum), Cloves (Syzygium aromaticum), ginger (Zingiber officinale) and turmeric (Curcuma longa) were evaluated in vitro against 17 Penicillium spp. Seed disease and rotten fruit caused by these species cause considerable loss of quality for different agricultural products. Isolates of Penicillium spp. were screened for production of patulin an important serious mycotoxin. About 70.59% of Penicillium spp. produced this toxin in concentrations ranging from 4 to 31 ppb. The response of Penicillium spp.to plant extracts differed according to the plant extract and concentration. Cinnamon extract showed the greatest effect on P. asperosporum, P. aurintogriseum and P. brevicompactum, and cloves extract produced the greatest effect on P. chermesinum and P. duclauxii. Turmeric extract had less effect on P. duclauxii. Cloves extract was the most effective in reducing the growth of Penicillium spp. On the other hand, ginger extract with all concentrations used had less effect against most Penicillium spp in the laboratory. Plant extracts are promising as natural sources of environmentally friendly compounds in laboratory studies.     

Biotechnological conversion of waste cooking olive oil into lipid-rich biomass using Aspergillus and Penicillium strains

Aims: In this study, we have investigated the biochemical behaviour of Aspergillus sp. (five strains) and Penicillium expansum (one strain) fungi cultivated on waste cooking olive oil. The production of lipid‐rich biomass was the main target of the work. In parallel, the biosynthesis of other extracellular metabolites (organic acids) and enzyme (lipase) and the substrate fatty acid specificity of the strains were studied. Methods and Results: Carbon‐limited cultures were performed on waste oil, added in the growth medium at 15 g l^?1, and high biomass quantities were produced (up to c. 18 g l^?1, conversion yield of c. 1·0 g of dry biomass formed per g of fat consumed or higher). Cellular lipids were accumulated in notable quantities in almost all cultures. Aspergillus sp. ATHUM 3482 accumulated lipid up to 64·0% (w/w) in dry fungal mass. In parallel, extracellular lipase activity was quantified, and it was revealed to be strain and fermentation time dependent, with a maximum quantity of 645 U ml^?1 being obtained by Aspergillus niger NRRL 363. Storage lipid content significantly decreased at the stationary growth phase. Some differences in the fatty acid composition of both cellular and residual lipids when compared with the initial substrate fat used were observed; in various cases, cellular lipids more saturated and enriched with arachidic acid were produced. Aspergillus strains produced oxalic acid up to 5·0 g l^?1. Conclusions: Aspergillus and Penicillium strains are able to convert waste cooking olive oil into high‐added‐value products. Significance and Impact of the Study: Increasing fatty wastes amounts are annually produced. The current study provided an alternative way of biovalourization of these materials, by using them as substrates, to produce added‐value compounds.     

Novel Antifungal and Antifeedant Metabolites from Penicillium chrysogenum Co-Cultured with Nemania primolutea and Aspergillus fumigatus

The endophyte Nemania primolutea, inhibited the growth of Penicillium chrysogenum in the coculture system. Four new compounds, nemmolutines A–B ( 1–2 ), and penigenumin ( 3 ) from N. primolutea, penemin ( 4 ) from P. chrysogenum were isolated from the coculture. On the other hand, P. chrysogenum inhibited the Aspergillus fumigatus in the coculture. Induced metabolites ( 13–16 ) with monasone naphthoquinone scaffolds including a new one from P. chrysogenum were produced by the coculture of P. chrysogenum, and A. fumigatus. Interesting, cryptic metabolites penicichrins A–B isolated from wild P. chrysogenum induced by host Ziziphus jujuba medium were also found in induced P. chrysogenum cultured in PDB ordinary medium. So the induction of penicichrin production by supplementing with host extract occurred in the fungus P. chrysogenum not the host medium. The productions of penicichrins were the spontaneous metabolism, and the metabolites ( 13–16 ) were the culture driven. Compounds 4 , 6 , 8 , 10 , 11 , 14 , and 15 showed significant antifungal activities against the phytopathogen Alternaria alternata with MIC_S of 1–8 μg/mL, and compounds 7 , 9 , and 12 indicated significant antifeedant activities against silkworms with feeding deterrence indexes (FDIs) of 92 %, 66 %, and 64 %. The carboxy group in 4-(2-hydroxybutynoxy)benzoic acid derivatives, and xylabisboeins; the hydroxy group in mellein derivatives; and the quinoid in monasone naphthoquinone increased the antifungal activities.     

曲霉生物膜的形成过程与结构特征

目的 研究曲霉生物膜的形成过程和结构特征.方法 我们利用一个曲霉生物膜体外模型研究其形成过程和结构特征.将200 μL浓度为1×10<'5>孢子/mL的受试曲霉(烟曲霉AF293,黄曲霉BMU03940,土曲霉BMU00802,黑曲霉BMU04689)的孢子悬液加到24孔组织培养板中的无菌塑料细胞培养盖玻片上,37℃孵育不同时间(0、2、4、8、10、12、16、18、24、48、72 h),加入25 μmol/L的FUN-1室温避光染色后,用波长488 nm激光激发,通过共聚焦激光扫描显微镜观察曲霉生物膜的形成过程;再用波长为488 am和633 am激光同时激发,将两个波长下的图像叠加后观察曲霉生物膜的活力;利用:XYZ轴成像观察其结构特征.在上述不同的时间点用钙荧光白染色后,用波长为405 nm的紫外光激发,观察曲霉生物膜细胞外基质的产生.结果 烟曲霉AF293在第4 h即开始有散在的孢子黏附于盖玻片上;8 h时孢子开始萌芽,10～12 h菌丝延长形成单细胞层;16～20 h菌丝缠绕形成多层立体结构;24 h形成一个具有复杂的三维立体结构特征的多细胞菌落,菌丝有序排列,细胞外基质弥散的分布在菌丝的周围;48～72 h生物膜逐渐成熟.成熟的烟曲霉生物膜是由细胞外基质包裹的有序排列的菌丝形成的复杂立体结构.黄曲霉BMU03940、土曲霉BMU00802、黑曲霉BMU04689与烟曲霉AF293有类似的生物膜发育阶段,包括黏附、孢子萌芽、菌丝延长、菌丝有序排列形成三维立体结构.结论 烟曲霉、黄曲霉、土曲霉和黑曲霉在体外都能形成典型的生物膜,它的形成过程和结构特征与其他真菌生物膜类似.     

致病性曲霉的耐药性研究进展

随着抗真菌药物在临床上的广泛使用,致病性真菌的耐药率越来越高,耐药曲霉对侵袭性曲霉病的诊治产生了重要影响。目前,致病性曲霉耐药性的确定主要依靠抗真菌药敏试验和分子诊断。在有关曲霉耐药机制的研究中,报道最多的是曲霉对唑类药物的耐药,其机制主要包括外排泵表达增加、靶酶Cyp51突变和表达水平增高、形成生物膜,以及热休克蛋白90（Hsp90）介导的信号通路参与而导致的耐药。本文就上述领域近年来的主要进展进行综述。