Mucor circinelloides - Springer Link

Mycotoxin Research Vol. 24, No. 3 (2008), 140-150

Detection of 3-nitropropionic acid and cytotoxicity in Mucor circinelloides M. Hollmann1, E. Razzazi-Fazeli1, J. Grajewski2, M. Twaruzek2, M. Sulyok3, J. Böhm1 1

University of Veterinary Medicine, Department of Farm Animals and Veterinary Public Health, Institute of Animal Nutrition, Veterinärplatz 1, 1210 Vienna, Austria 2 Kazimierz Wielki University, Department of Experimental Biology, Division of Physiology and Toxicology, Chodkiewicza 30, 85064 Bydgoszcz, Poland 3 University of Natural Resources and Applied Life Sciences Vienna, Department IFA-Tulln, Konrad-Lorenz-Strasse 20, 3420 Tulln, Austria

Abstract

Mucorales are regarded as the aetiological agents of Mucormycosis. Their capabilities to produce mycotoxins are not profoundly investigated, in contrast to those of the fungi from the genera Penicillium, Aspergillus, or Fusarium. The aim of this study was to isolate and identify fungi of the order Mucorales and investigate mycotoxins production. Twelve samples of visibly moulded grass silage and eight samples of damaged whole crop maize silage were analysed. Malt extract agar plates were used for sub cultivation. Three fungal species of the order Mucorales were isolated from grass silage, which were identified by their macro- and micromorphology as Absidia corymbifera, Mucor circinelloides and Rhizopus stolonifer. The cytotoxicity of Mucor circinelloides extract was analysed using the cytotoxicity test (MTT assay) and the result showed a low cytotoxicity. Additionally extracts from Absidia corymbifera, Mucor circinelloides and Rhizopus stolonifer were tested for mycotoxinproduction using an LC/MS/MS-based multimycotoxin method. 3-nitropropionic acid was detected in the culture extract of Mucor circinelloides. Keywords: mucorales, silage, cytotoxicity, 3-nitropropionic acid Introduction

Mucorales is the largest group of the Zygomycetes, encompassing such important genera as Absidia, Mucor and Rhizopus. These are mostly fast-growing fungi and are saprophytic microorganisms. Mycelial elements expand and cover the entire plate in only a few days. The mycelium is described as “cotton candylike”. At the sexual form zygospores are produced and sporangiospores are produced in sporangia occur during asexual reproduction (1). Some Mucor spp. as well as Rhizopus spp. are important as fermentation agents for food (for example tempeh) and are used extensively in the biotechnology for enzyme and organic acid production (2, 3, 4). The Mucorales are widespread in nature and can be found on food, grain, vegetables, fruits, nuts and silage (5, 6). The taxonomy of this group of fungi is undergoing massive changes. Figure 1 shows the taxonomic th

Presented at the 30 Mykotoxin Workshop Utrecht, Netherlands, April 28-30, 2008 Correspondence: Manfred Hollmann, University of Veterinary Medicine, Department for Farm Animals and Veterinary Public Health, Institute of Animal Nutrition, Veterinärplatz 1, 1210 Vienna, Austria ([email protected]) Received 15 Oct 2008; accepted 14 Nov 2008

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organisation of the Zygomycetes. Hibbett et al. (7) suggested a new subphylum Mucormycotina, which included the order of Mucorales. Mucorales are regarded as the aetiological agents of mucormycosis, which is the third most common invasive fungal infection after aspergillosis and candidiasis (8, 9, 10, 11). The most common species causing Mucormycosis are R. arrhizus, R. microsporus var. rhizopodiformis, A. corymbifera and Mucor spp. (9, 11, 12). Mucormycosis is an infection in immunosuppressed patients. The hallmark of Mucormycosis is vascular invasion resulting in thrombosis and infarction of tissue (13, 14, 15,). The risk factors of Mucormycosis are diabetes mellitus, diabetic ketoacidosis, haematologic malignancy, neutropenia, organ transplantation, deferoxamine therapy, trauma and burns (8, 12, 16, 17). Clinical diagnosis of Mucormycosis is difficult, and is often carried out at a late stage of the disease or post-mortem (10). The major clinical manifestations of Mucormycosis are rhinocerebral, pulmonary, cutaneous, gastrointestinal and disseminated. The incidence of infectious diseases caused by Mucormycosis has risen significantly over the past decade (12). The overall rate of mortality of Mucormycosis is approximately 40% (10).


Kingdom:

Fungi

Phylum: Ascomycota

Basidiomycota

Class:

“Mitosporic fungi” (“Fungi imperfecti”)

Zygomycetes

Order:

Family: Genus: Species:

Zygomycota

Mucorales

Mucoraceae Absidia A. corymbifera Apophysomyces A. elegans Mucor M. circinelloides M. hiemalis M. racemosus M. ramossimus M. rouxianus Rhizomucor R. pusillus R. miehei Rhizopus R. arrhizus R. azygosporus R. microsporus Var. microsporus Var. oligosporus Var. rhizopodiformis R. schipperae R. stolonifer

Figure 1. Taxonomic organization of the zygomycetes (Ribes et al., 2000, modified)

Table 1. Indicator germ group (cfu/g) and assessment of the mycological flora of silage (VDLUFA, 2004; Richter, 2008) Group

Importance

Germ Group

Anerobic mesophilic bacteria

Product- typical

KG 1 KG 2

Spoil-indicating KG 3

Indicator germ Yellow germ Pseudomonas, Enterobacteriaceae Other product-typical bacteria Bacillus spp. Staphylococcus, Micrococcus Streptomyceten

Corn Silage

Grass Silage

5 x 105

2 x 106

3 x 10 3 x 104

5

3 x 10 3 x 104

5 x 103

1 x 104

1 x 104

1 x 10

5

Black fungi Verticillium spp.

Product- typical KG 4 Mould and black fungi

Acremonium spp. Fusarium spp. Auerobasidium spp. Other product-typical fungi Aspergillus spp.

Spoil-indicating

KG 5

Penicillium spp. Scopulariopsis spp. Wallemia spp.

4

Other spoil-indicating fungi KG 6 Yeasts

Spoil-indicating

KG 7

Mucorales All genera

141

3

5 x 103

6

5 x 105

3 x 10 1 x 10


In contrast, pulmonary mucormycosis has a high mortality (65%) and in patients with disseminated mucormycosis the mortality approaches 100% (14). Additionally, mucormycosis has been diagnosed in many animal species, including marine mammals, cattle, sheep, swine, horse, tree frogs and birds (16, 17, 18, 19, 20, 21, 22). The VDLUFA (Verband Deutscher Landwirtschaftlicher Untersuchungs- und Forschungsanstalten) has modified the order Mucorales in an own germ group (Table 1). Mucorales proliferate at higher humidity and produce late sporangia. In contrast to Aspergillus spp., Penicillium spp. and other mould Mucorales produce fewer spores higher part of mycelium. Table 1 shows the assessment of the mycological flora of silage with provisional recommended value forage (23). The order Mucorales apparently contains few mycotoxin producers, which are not excluded from potential mycotoxin producers. However, compounds of sufficient toxicity to be termed mycotoxins have not been found in these genera (24). The information in the literature about Mucorales and their mycotoxins is rare. Some authors reported about the capacity of Mucorales to produce mycotoxins such as aflatoxin (25), ergot alkaloid (26, 27, 28), agroclavine (29) and fumigaclovine (30). Table 2 gives an overview of the available literature. About two decades ago rhizozin A and B and rhizoxin were identified from highly toxinogenic Rhizopus microsporus strain (31). The rhizonins A and B have a strong hepatotoxic activity and can cause a wide range of hepatic lesions (31, 32). Rhizoxin has been shown to have antimicrobial, antifungal activities (31). Additionally in vitro cytotoxicity and in vivo antitumor activity have been described (31). However,

rhizonin and rhizoxin is in fact not produced by the fungus, but is produced by bacteria (Burkholderia spp.) that reside within the fungal cytosol (32). Rhizopus spp. produces a variety of enzymes, proteins and by-products that have pathogenic potential (5). The aim of this study was to isolate and identify fungi of the order Mucorales and investigate mycotoxins production in low quality grass silage and whole crop maize silage.

Materials and Methods

Twenty silage samples (12 grass silages and 8 whole crop maize silages), which showed visible fungal contamination (103-107cfu/g), were analysed. For each sample, 20 g of silage was suspended in 380 ml sterile water. After 20 minutes of shaking, serial dilutions were up to 10-2 prepared. Standard cultivation media, sabouroud-2% glucose agar (Oxoid, England), malt extract agar (MEA, Oxoid, England) and Czapek yeast extract agar (CYA, Oxoid, England) were used. Aliquots of 1 ml of each dilution were plated on MEA plastic petri dishes. All petri dishes were incubated in the dark at 25 °C for 7 days. The total number of mould and yeast by each silage sample were counted and their numbers were expressed as colony forming units per gram (cfu/g). The identified mucorales were subcultivated on MEA, CYA and sabouroud-2% glucose agar by different temperatures (25 °C, 37 °C, 45 °C). For characterisation a microscope slide and a transparent adhesive tape were used. The colonies are lightly touched with the adhesive tape. After this the tape is transferred to a microscope slide with a drop of mounting medium (lacto phenol cotton blue, Merck, Germany).

Table 2. Mucorales and their reported mycotoxins Mucorales

Toxin

References

Mucor mucedo Fr. Rhizopus nigricans Ehrenb. Mucor hiemalis W. Mucor hiemalis Rhizopus nigricans Rhizopus nigricans Rhizopus arrhizus Rhizopus arrhizus Rhizopus arrhizus Rhizopus stolonifer (Ehrenb. Ex Fr.) Vuill.

Aflatoxin Aflatoxin Ergot alkaloid Ergot alkaloid Agroclavine Ergot alkaloid Agroclavine Fumigaclovine B Ergot alkaloid Ergot alkaloid

LOIVEKE et al., 2004 (25) LOIVEKE et al., 2004 (25) LUGAUSKAS et al., 2005 (26) NIELSEN et al., 2004 (27) FLIEGER et al., 1997 (29) NIELSEN et al. 2004 (27) FLIEGER et al., 1997 (29) COLE et al., 2003 (30) NIELSEN et al. 2004 (27) KRISTAPONIS et al. 2001 (28)

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Mucorales were identified to genera and species by their macro- and micro morphology features using appropriate identification keys according to Samson et al., Liou et al., Shipper et al. and Seeliger et al. (6, 33, 34, 35). All pictures of the fungi were produced with a microscope (Polyvar, Reichert-Jung, Austria) and a digital net camera (DN100, Nikon, Germany).

tetrazolium salt MTT to a photometrically measurable purple formazan derivative by mitochondrial enzymes of metabolically active cells (36, 37). The fungus M. circinelloides was extracted 3x with 0.5 ml chloroform (SigmaAldrich, Austria) directly from the MEA-petri dish, after drying at 100 °C over night. The extract of M. circinelloides was filtered through 0.22 μm filters (Sartorius, German). Swine kidney monolayers cells (SK) derived from an adherent cell line with epitheloid morphology, and were used as target cells. These target cells have a proven sensitivity for a wide range of mycotoxins. The extract of M. circinelloides was dissolved in a mixture of ethanol-

MTT-assay The MTT-cell culture assay used to determine cytotoxicity of potential medicinal agent and other toxic materials. This MTT-assay is based on the conversion of the yellow

Table 3. The106 metabolites analysed by LC-ESI-MS/MS (40) 3-Acetyldeoxynivalenol

Elymoclavine

Mevinolin

15-Acetyldeoxynivalenol

Elymoclavine-fructoide

Moniliformin

AAL TA-Toxin

Emodin

Monoacetoxyscirpenol

Aflatoxin B1

Enniatin A

Mycophenolic acid

Aflatoxin B2

Enniatin A1

Neosolaniol

Aflatoxin G1

Enniatin B

3-Nitropropionic acid

Aflatoxin G2

Enniatin B1

Nivalenol

Aflatoxin M1

Enniatin B3

Ochratoxin A

Agroclavine

Ergine

Ochratoxin B

Alamethicin F30

Ergocornine

Ochratoxin

Altenuene

Ergocorninine

Oxidized elymoclavine

Alternariol

Ergocristine

Oxidized luol

Alternariolmethylether

Ergocristinine

Patulin

2-Amino-14,16-dimethyloctadecan-3-ol

-Ergocryptine

Paxilline

Avenacein Y

-Ergocryptinine

Penicillic acid

Beauvericin

Ergometrine

Penitrem A

Brefeldin A

Ergometrinine

Roquefortine C

Chaetoglobosin A

Ergosine

Roridin A

Chanoclavine

Ergosinine

Sterigmatocyctin

Chetomin

Ergotamine

Sulochrin

Citrinin

Ergotaminine

T2-tetraol

Cytochalasin B

Festuclavine

T-2 toxin

Cytochalasin D

Fumitremorgin C

T2-triol

Cytochalasin E

Fumonisin B1

Tentoxin

Cytochalasin H

Fumonisin B2

Verrucarin A

Cytochalasin J

Fumonisin B3

Verrucarol

Cytocholasin A

Fusareon-X

Verruculogen

Cytocholasin C

Gibberellic acid

Zearalenone

Cytocholasin C

Gliotoxin

Zearalenone-4-glucoside

Deepoxydeoxynivalenol

Griseofulvin

Zearalenone-4-sulfate

Deoxynivalenol

HC-toxin

-Zearalenol

Deoxyniv.-3-glucosid

Hydrolyzed fumonisin B1

-Zearalenol

Diacetoxyscirpenol

Kojic acid

-Zearalenol-glucosid

Dihydroergosine

Lysergol

-Zearalenol-glucosid

Dihydroergotamine

Meleagrin

Dihydrolysergol

Mathysergide

143


dimethylsulfoxide-minimum essential medium with Earle's salts (MEM) (1.7 + 0.3 + 98, v/v/v) as described by Hanelt et al. (38). Serial log 2 dilutions of the sample extract were made. All plates were incubated for 48 hours at 37 ºC in a humidified atmosphere with 5% CO2. A volume of 20 l of the MTT stock solution was then added to each wells and plates incubated for another 4 hours. Supernatant was then removed using a multichannel micropipette and 100 l DMSO was added to each well measured spectrophotometrically with an ELISA-Reader. The absorbance was measured at 510 nm, the wavelength of maximum absorption of the formazan derivative. The cytotoxicity was defined as the lowest concentration of a sample extract to cause reduced MTTcleavage activity (mg of sample aliquot per ml cell culture medium) - low value-high cytotoxicity. The limit for significant cell toxic response was set to 50% metabolic activity compared to cell control, as this was the highest percentage for which a linear concentration/response curve was obtained.

shows a retention time of 2.93 min and limit of detection was found to be 25 μg/kg. Results

Table 4 shows the total number of counted mould and yeast from the silage samples (cfu/g of silage). Three fungal species of the order Mucorales were isolated from grass silage and were identified by their macro- and micromorphology as Absidia corymbifera (Figure 2 and 3), Mucor circinelloides (Figure 4) and Rhizopus stolonifer (Figure 5 and 6). A. corymbifera was isolated from grass silage number 3, M. circinelloides from grass silage number 10 and R. stolonifer from grass silage number 11. In contrast Mucorales could not be isolated from maize silage. MTT-assay The results of the MTT-assay are shown in Table 5; as can be seen M. circinelloides demonstrate low cytotoxicity. Additionally a control sample was parallel carried out being negative.

LC-ESI-MS/MS Liquid chromatography with electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS) was used for mycotoxin detection according to Sulyok et al. (39, 40). This multi-mycotoxin method is able to quantify 106 mycotoxins (Table 3). For the extraction a mixture of acetonitrile (SigmaAldrich, Austria), water and acetic acid (Sigma-Aldrich, Austria) was used. A volume of 4 ml of extraction solvent of acetonitrile/water/acetic acid (79/20/1 v/v/v) was added to one gram of brayed fungi culture in a mortar. The extracts were filtered through 0.45 μm filters and transferred to clean vials. The extracts from A. corymbifera, M. circinelloides and R. stolonifer were analysed by LCESI-MS/MS. For detection and quantification of 3-Nitropropionic acid m/z 118 as the precursor ions >M-H@- and m/z 46 as product ions were taken. The declustering potential and collision energy were set to -35 and -16 V respectively with a dwell time of 100 ms. In chromatographic system 3-nitropropionic acid

Table 4. Total number of counted yeast and mould (cfu/g)

144

Sample

Culture media

yeast

mould

1

Grass silage

Sabouraud

7,1E+06

7,0E+04

2

Grass silage

Sabouraud

1,0E+06

2,0E+04

3

Grass silage

MEA

>3,0E+07

2,0E+05

4

Maize silage

MEA

>3,0E+07

1,50E+07

5

Grass silage

MEA

1,50E+07

3,00E+06

6

Maize silage

MEA

>3,0E+07

2,20E+06

7

Maize silage

MEA

>3,0E+07

2,00E+04

8

Grass silage

MEA

3,00E+05

0,00E+00

9

Maize silage

MEA

1,00E+07

2,80E+04

10

Grass silage

MEA

>3,0E+07

1,00E+07

11

Grass silage

MEA

1,00E+07

9,00E+05

12

Grass silage

MEA

1,00E+07

9,00E+05

13

Grass silage

MEA

>3,0E+07

8,00E+05

14

Grass silage

MEA

>3,0E+07

5,00E+05

15

Maize silage

MEA

>3,0E+07

0,00E+00

16

Grass silage

MEA

6,00E+06

0,00E+00

17

Maize silage

MEA

3,80E+06

1,00E+03

18

Maize silage

MEA

>3,0E+07

2,00E+05

19

Grass silage

MEA

1,00E+07

1,00E+05

20

Maize silage

MEA

>3,0E+07

2,00E+04


Sporangium

Sporangiophore

40μm

Figure 2. Absidia corymbifera

(from grass silage 3)

Apophysis

Sporangiospores

60 μm

Figure 3. Absidia corymbifera (from grass silage 3)

Columella

Sporangiospores

Sporangiophores 100 μm

Figure 4. Mucor circinelloides (from grass silage 10)

145


Columella

Sporangiospores

Sporangiophore 100 μm

Figure 5. Rhizopus stolonifer (from grass silage 11)

Columella Rhizoids

Sporangiophore

100 μm

Figure 6. Rhizopus stolonifer (from grass silage 11)

Table 5. MTT-assay of Mucor circinelloides

Sample

Kind of sample

Step

IC 50 [cm²/ml]

Cytotoxicity

MEA

MEA Medium - control

-

>31,25

control

MEA Medium, dried in 100 ºC; MEA (dried) 3x0,5 ml chloroform, filtred PVDF, 0,45μm - control

1

31,25

control

Mucor circinelloides

2

15,61

(+) Low cytotoxic

Sample

146


2.8e4 2.6e4

Peak at 3.01 min 3-nitropropionic acid

2.4e4 2.2e4 2.0e4

Intensity

1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 8000.0

2.81

6000.0 4000.0 6.44 3.65

2000.0 0.0

4.66

14.69

7.32 7.88 10.87

8.638.84 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Time, min

Figure 7. LC-ESI-MS/MS SRM Chromatogram of an extract of Mucor circinelloides

LC-ESI-MS/MS Extracts of A. corymbifera, M. circinelloides and R. stolonifer were analysed by the LCESI-MS/MS. 3-nitropropionic acid was found only in the extract of M. circinelloides (Figure 7). The concentration of 3-nitropropionic acid in this extract was found to be 816 ng/g. In contrast in the extracts of A. corymbifera and R. stolonifer were none of the 106 mycotoxins detected.

toxicity of fungi against larvae of the brine shrimp and chicken embryos and found M. fragilis and R. nigricans (R. stolonifer) to be toxic (48). Makun et al. isolated fungi from millet; and extracts of these fungi were tested on mice (49). Each mouse received 0.2 ml of cotton-seed oil extract of fungi culture by intraperitonial injection. The results show that R. stolonifer were moderately toxic (49). In addition the species of the order Mucorales A. butleri, A. glauca, M. circinelloides, M. griseocyanus, M. mucedo and R. stolonifer have been reported to produce aflatoxins, but this is incorrect (24). In this study, we could observe low cytotoxicity of Mucor circinelloides. Additionally, the analysis of mycotoxins revealed that Mucor circinelloides is able to produce 3nitropropionic acid. 3-nitropropionic acid has been reported to be produced by some genera of the Leguminosae family and the moulds Arthrinium aureum, A. phaerospemum, A. sacchari A. saccharicola, A. sereanis, A. terminalis. Aspergillus flavus, A. oryzae, A. sojae, Penicillium atrovenetum (50, 51, 52, 53, 54, 55, 56).

Discussion

The purpose of this study was to isolate and identify fungi of the order Mucorales and find out which mycotoxins are produced by these species. Mucorales have been reported to be often isolated from silage (41, 42, 43, 44, 45, 46, 47) and are regarded as the aetiological agents of Mucormycosis. However, their capabilities to produce mycotoxins have not been profoundly investigated and little information is available on toxin production by Mucorales. Davis et al. screened the

147


References

The primary mechanism of 3-nitropropionic acid has been investigated to be a noncompetitive inhibitor of succinat dehydrogenase, an enzyme of the mitochondrial membrane that catalyzes the oxidation of succinat to fumarat (57, 59). The clinical picture consists of an acute encephalopathy followed by appearance of a delayed onset dystonia after ingestion, with symptoms resembling those of Huntington disease (58, 59). Human intoxication of 3-nitropropionic acid has occurred in China via ingestion of Arthrinium spp. contaminated sugarcane (53). Anderson et al. have found that 3-nitropropionic acid is metabolized by the ruminal microbes or rapidly absorbed in the reticulo-rumen (50). When given intravenously, 3-nitropropionic acid was lethal to sheep in a concentration of 52 mg/kg body weight (50). This paper is the first to report on the ability of M. circinelloides for production of 3-nitropropionic acid in culture. However, there are rare reports on the occurrence of 3-nitropropionic acid in food and feedstuffs.

1 Reiss J (1997) Schimmelpilze Lebensweisen Nutzen Schaden Bekämpfung. 2. Auflage, Springer-Verlag, Berlin, 1-33 2 Jennessen J, Fog Nielsen K, Houbraken J, Lyhne EK, Schnürer J, Frisvad JC, Samson RA (2005) Secondary Metabolite and Mycotoxin Production by the Rhizopus microsporus Group. J Agric Food Chem 53: 1833-1840 3 Campbell-Platt G, Cook PE (1989) Fungi in the production of foods and food ingredients. Journal of Applied Bacteriology Symposium 18 (Suppl.): 117-131 4 Spök A, Proksch M (2007) Lebensmittelenzyme in der EU Herstellung, Anwendung, Marktsituation und Rechtliche Regelungen. Forschungsberichte der Sektion 4, Band 3 5 Ribes JA, Vanover-Sams CL, Baker D (2000) Zygomycetes in Human Disease. Clin Microbiol Rev 13 (2): 236-301 6 Samson RA, Hoekstra ES, Frisvad JC, Filtenborg O (2000) Introduction to Food- and Airborne Fungi. Centraalbureau voor Schimmelcultures, Utrecht, 1-25 7 Hibbett DS, Binder M, Bischoff JF, Blackwell M, Cannon PF et al. (2007) A higer- level phylogenetic classification of the fungi. Mycol Res 3: 509-547 8 Prabhu RM, Patel R (2004) Mucormycosis and entomophthoramycosis: a review of the clinical manifestation, diagnosis and treatment. Clin Microbiol Infect 10 (Suppl. 1): 31-47 9 Eucker J, Sezer O, Graf B, Possinger K (2001) Mucormycoses. Mycoses 44: 253-260 10 Bouza E, Munoz P, Guinea J (2006) Mucormycosis: an emerging disease? Clin Microbiol Infect 12 (Suppl. 7): 7-23 11 Richardson M, Lass-Flörl C (2008) Changing epidemiology of systemic fungal infections. Clin Microbiol Infect 14 (Suppl. 4): 5-24 12 Chayakulkeeree M, Ghannoum A, Perfekt JR (2006) Zygomycosis: the re-emerging fungal infection. Eur J Clin Microbiol Infect Dis 25: 215229 13 Weitzman I, Della-Latta P (1997) Emerging Zygomycotic Agents. Clinical Microbiology Newsletter 19 (11): 81-88 14 Spellberg B, Edwards J, Ibrahim A (2005) Novel perspectives on mucormycosis: pathophysiology, presentation, and management. Clin Microbiol Rev 18 (3): 556-569 15 Roden MM, Zautis TE, Buchanan WL, Kundsen TA, Sarkisova TA, Saufele RL, Sein M, Sein T, Chiou CC, Kontoyiannis DP, Walsh TJ (2005) Epidemiology and outcome of Zygomycosis: A review of 929 reported cases. Clin Infect Dis 41: 634-653

Conclusions

Mucorales were often isolated from food and feed. However, no information was available on their ability to produce mycotoxins. We have found 3 species of Mucorales in damaged grass silage and M. circinelloides was found to produce 3-nitropropionic acid and demonstrates a low cytotoxicity. In contrast in the extracts of A. corymbifera and R. stolonifer no mycotoxins could be detected. Silage can often obtain Absidia spp., Mucor spp. and Rhizopus spp. which could cause mucormycosis in humans or animals. However, further studies are needed to examine, if M. circinelloides spoiled silage are risky feed for ruminants.

Acknowledgements

The authors would like to express their gratitude to Anna Blajet-Kosicka and Beata Miklaszewska from Kazimierz Wielki University (Poland) for carrying out the MTT-assay and verify 3-nitropropionic acid and their assistance in identification of fungi.

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