ilable at ScienceDirect

Toxicon 53 (2009) 283–288

Contents lists ava

Toxicon

journal homepage: www.elsevier .com/locate/ toxicon

Mycological survey and ochratoxin A natural contamination of swinefeedstuffs in Rio de Janeiro State, Brazil

C.A.R. Rosa a,b, K.M. Keller a,c, L.A.M. Keller a,c, M.L. Gonzalez Pereyra d,e, C.M. Pereyra d,e,A.M. Dalcero d,f, L.R. Cavaglieri d,f,*, C.W.G. Lopes a

a Departamento de Microbiologia e Imunologia Veterinaria, Universidade Federal Rural do Rio de Janeiro, Instituto de Veterinaria, Rodovia BR 465 Km 7,Seropedica 23890-000, Rio de Janeiro, Brazilb Member of Conselho Nacional de Pesquisas Cientıficas (CNPq), Brazilc Fellow of Conselho Nacional de Pesquisas Cientıficas (CNPq), Brazild Departamento de Microbiologıa e Inmunologıa, Universidad Nacional de Rıo Cuarto, Ruta 36 Km 601, (5800) Rıo Cuarto, Cordoba, Argentinae Fellow of Consejo Nacional de Investigaciones Cientıficas y Tecnicas (CONICET), Argentinaf Member of Consejo Nacional de Investigaciones Cientıficas y Tecnologicas (CONICET), Argentina

a r t i c l e i n f o

Article history:Received 22 September 2008Received in revised form 27 November 2008Accepted 28 November 2008Available online 6 December 2008

Keywords:CornBrewers’ grainsFeedsFungiMycotoxins

* Corresponding author. Departamento de Microgıa, Universidad Nacional de Rıo Cuarto, Ruta 36Cuarto, Cordoba, Argentina. Tel.: þ54 358 46764676231.

E-mail address: lcavaglieri@arnet.com.ar (L.R. Ca

0041-0101/$ – see front matter � 2008 Elsevier Ltddoi:10.1016/j.toxicon.2008.11.015

a b s t r a c t

Mycotoxin contamination of animal feeds represents a hazard to human and animal healthdue to potential transmission to meat and milk. Barley by-products are alternative feedingsupplies for animal production. The aims of this assay were to study the mycobiota offeedstuffs and finished swine feed, to determine the ability of Aspergillus and Penicilliumisolates to produce ochratoxin A (OTA) and to evaluate OTA occurrence in these substrates.Corn, brewers’ grains and finished swine feed samples were collected from differentfactories. Fungal counts were higher than 2.8� 104 CFU g�1. Fusarium, Aspergillus andPenicillium genera were isolated at high levels. A 23.7% of the isolates produced9–116 mg kg�1 of OTA in vitro. Corn samples (44%) were contaminated with 42–224 mg kg�1

of OTA. Finished feed (31%) and brewers’ grains samples (13%) were contaminated with36–120 mg kg�1 and 28–139 mg kg�1 of OTA, respectively. This is the first scientific reporton contamination by OTA-producer molds and OTA in swine feedstuffs from Brazil. Thepresence of OTA in raw materials and finished feed requires periodic monitoring to preventmycotoxicoses in animal production, reduce economic losses and minimize hazards tohuman health.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction may also occur during processing and storage of harvested

Mycotoxin contamination of animal feeds representsa serious hazard to human and animal health due thepotential transmission of the toxins to meat, milk and by-products. Mycotoxin contamination of feed ingredientsfrequently occurs in the field when plants are infected byparticular pathogenic fungi or by symbiotic endophytes. It

biologıa e Inmunolo-Km 601, (5800) Rıo429; fax: þ54 358

vaglieri).

. All rights reserved.

products and feeds whenever environmental conditionsare appropriate for spoilage fungi development (D’Melloand Macdonald, 1998). Barley and by-products are aninteresting alternative option as feeding supplies for animalproduction, being a rich source of protein and fiber at a lowprice (Lopez-Diaz and Flannigan, 1997; Fagundes, 2003).However, further study is needed to know their influenceon fungal and ochratoxin A (OTA) contamination of thefinal feed. Aspergillus spp., Penicillium spp. and Fusariumspp. are the most commonly filamentous molds found instored cereal grains and feeds. They can cause foodspoilage, biodeterioration and are able to produce differentmycotoxins. Ochratoxin A is one of the most common and

C.A.R. Rosa et al. / Toxicon 53 (2009) 283–288284

dangerous mycotoxins in foods and feeds, naturallyproduced by Aspergillus ochraceus, Aspergillus carbonariusand Aspergillus niger aggregate mainly in tropical regions,and Penicillium verrucosum in temperate areas (VanEgmond and Speijers, 1994; Teren et al., 1996; Abarca et al.,2001; Magnoli et al., 2004, 2006a,b). Interest on OTAincreased when it was classified as 2B possible humancarcinogen by the International Agency for Research onCancer, based on sufficient evidence of carcinogenicity inexperimental studies on animal (IARC, 1993). Ochratoxin Ahas a potent toxicity and its nephrotoxic, hepatotoxic,teratogenic, carcinogenic and immunosuppressive effectshave been demonstrated in many mammalian species(Karlovsky, 1999). The use of OTA contaminated feed duringa long rearing period involves serious risk due to thereduction of feed efficiency with a subsequent growthdecrease and immune system weakening. OTA has beenimplicated in several mycotoxicoses in farm livestock inItaly, Israel, Denmark and Brazil (Krogh, 1978; Visconti andBottalico, 1983; Cruz et al., 1984; Shlosberg et al., 1997). Inprevious studies we have determined the mycobiota andOTA contamination from poultry and cow’s feeds in Brazil(Rosa et al., 2006, 2008). However, few data are available onthe ochratoxigenic mycobiota and OTA production in swinefeedstuffs. The aims of this study were: i) to determine themycobiota present in feedstuffs and finished swine feed,ii) to determine the ability of several Aspergillus and Peni-cillium isolated strains to produce OTA and, iii) to evaluatethe occurrence of OTA in these substrates.

2. Materials and methods

2.1. Source of samples

A total of 74 samples including corn (32) and brewers’grains (16) – used as ingredients for feed manufacturing –as well as finished swine feed samples (26) were collectedfrom August 2005 to July 2006, from different factorieslocated in Rio de Janeiro State, Brazil. These primarysamples were homogenized, quartered to get 2.5 kg labo-ratory samples and tested immediately for water activity(aW), total mold counts, fungal isolation frequency andrelative density. Then, they were stored at 4 �C untilmycotoxin analysis.

2.2. Water activity of swine feeds

Water activity (aW) determinations were carried outusing AQUALAB CX2 (Decagon, Devices, Inc., USA). Thedevise was calibrated by appropriate saline solutions in thework interval – according to procedures manual – beforesample measurements.

2.3. Mycobiota determination

Quantitative enumeration of fungal propagules wasdone in a solid medium using the surface-spread method.Ten grams of each sample were homogenized in 90 ml 0.1%peptone water solution for 30 min. Serial dilutions 10�2 to10�4 were made and 0.1 ml aliquots were inoculated intriplicates onto dichloran rose bengal chloranphenicol agar

(DRBC) – used for general fungal enumeration – anddichloran 18% glycerol agar (DG18) – for xerophilic fungi(Pitt and Hocking, 1997). Plates were incubated at 28 �C for7 days. Plates containing 10–100 colony forming units(CFU) were used for total fungal counts and the resultswere expressed as CFU per gram of sample. One colony ofeach type considered to be different isolated from eachsample was transferred to malt extract agar (MEA) andincubated at 28 �C for 7 days for latter identification.Taxonomic identification of the different genera andspecies was done according to Pitt and Hocking (1997)and Klich (2002). The isolation frequency of each genus andrelative density of each species were calculated as follows(Castillo et al., 2004):

F ¼�ns

N

�� 100 and RDð%Þ ¼

�niNi

�� 100

where F¼ isolation frequency; ns¼ number of samples inwhich a certain genus occurred; RD¼ relative density;N¼ total number of samples; ni¼ number of isolates ofa certain genus; Ni¼ total number of fungal isolates obtained.

2.4. Ochratoxin A production

The OTA production was tested in 317 strains belongingto Nigri (A. niger aggregate and A. carbonarius), Circundati(A. ochraceus and Aspergillus melleus), Terrei sections(Aspergillus terreus) and strains of P. verrucosum. OchratoxinA was determined according to the methodology describedby Teren et al. (1996). The strains were grown in stationarycultures in 125 ml Erlenmeyer flasks containing 30 ml ofYES culture medium (2% yeast extract, 15% sucrose). Denseconidial suspensions (100 ml) of each strain were inoculatedinto the flasks and incubated at 30 �C for 30 days in thedark. Aliquots (1 ml) of these cultures were then mixedwith 1 ml chloroform and centrifuged at 4000g � for10 min. The chloroform phase was transferred to a cleanvial, evaporated to dryness and re-dissolved in 0.5 mlmethanol. The extracts were stored at �20 �C untilanalyzed. The production of OTA was detected by HPLCfollowing the methodology subsequently described.

2.5. Ochratoxin detection and quantification

Ochratoxin A analysis in swine feedstuff samples wascarried out by HPLC following the methodology proposed byScudamore and MacDonald (1998), with some modifications.A 50 g portion of each sample was extracted with meth-anol:water solution (80:20, v/v) over 30 min in an orbitalshaker. The mixture was filtered to remove particulate matterand a 10-ml aliquot was taken and diluted with 40 ml distilledwater. Ten milliliters of the latter were added to an immu-noaffinity column (OchraTest�, Vicam, Digen, Oxford, UK).The column was washed twice with 10 ml PBS containing0.01% Tween 20 and then with 10 ml double-distilled water,respectively. Ochratoxin A was eluted from the column withHPLC-grade methanol at one to two drops per second flowrate. The extract was dried under nitrogen flow. The driedextracts were re-dissolved in 500 ml chloroform and detectedon an HPLC system. The HPLC device used for OTA

Fig. 1. Total fungal counts (CFU g�1) of swine feedstuffs. Hygienic qualitylimits (1.0�104 CFU g�1) were determined according to GMP (2008).Different letters (a and b) indicate statistically significant difference(P< 0.05).

Fig. 2. Isolation frequency (%) of different genera isolated from swinefeedstuffs.

C.A.R. Rosa et al. / Toxicon 53 (2009) 283–288 285

determination was a Hewlett Packard chromatograph witha 20 ml loop, equipped with a spectrofluorescence detector(excitation l, 330 nm; emission l, 460 nm) and a C18 column(Supelcosil LC-ABZ, Supelco; 150 mm� 4.6 mm, 5 mm particlesize) connected to a precolumn (Supelguard LC-ABZ, Supelco;20 mm� 4.6 mm, 5 mm particle size). The mobile phase waspumped at 1.0 ml min�1 and consisted of an isocratic systemas follows: 57% acetonitrile, 41% water and 2% acetic acid. OTAwas quantified on the basis of fluorometric responsecompared with OTA standard solution (Sigma-Aldrich,St. Louis, MO, USA; purity >99%). The detection limit of thetechnique was 0.4 mg kg�1.

2.6. Spiking and recovery assay

A stock solution (4 ml of 5 mg ml�1 OTA in methanol)was prepared for recovery determination. OTA-free feedsamples (50 g each) contained in 250 ml Erlenmeyer flaskswere spiked with equivalents to 10, 20 and 40 mg OTA kg�1.Spiking was done in triplicates and a single analysis of theblank sample was carried out. After 16 h (after methanolevaporation), extraction solvent was added and OTAconcentration was determined using the previouslydescribed protocol. The recovery rates� standard error ofthe methodology (n¼ 3) were 87� 5%, 84� 5% and 86� 5%for OTA, respectively.

2.7. Statistical analysis

Data analyses were performed by analysis of variance.Total fungal counts data were transformed using a loga-rithmical function log10(xþ 1) before applying the analysisof variance. The LSD test was used to determine thesignificant differences between means. The analysis wasconducted using PROC GLM in SAS (SAS Institute, Cary, NC).

3. Results

3.1. Water activity content

The highest water activity values were registered inbrewers’ grains samples with a mean of 0.936� 0.082. Thecorn kernels and finished swine feed samples tested had aW

mean values of 0.627� 0.112 and 0.628� 0.055, respectively.

3.2. Mycological isolation and identification

Total fungal counts from all analyzed samples are shownin Fig. 1. In general, all samples showed counts higher than2.8� 104 CFC g�1. Counts on DG18 and DRBC were similar,except for corn samples that had showed lower moldcounts on DG18 (P< 0.05). Fig. 2 shows the mold generaisolation frequency from all samples. Fusarium spp. was theprevalent genera (100%), followed by Penicillium spp. andAspergillus spp. Other genera were isolated at lowerfrequencies. Brewers’ grains samples showed the highestyeast contamination (data not shown). Fig. 3 showsAspergillus spp. relative densities. Aspergillus flavus was thepredominant species (51%) followed by A. niger aggregate(21%) and Aspergillus parasiticus (15%). Aspergillus sectionFlavi obtained 66% of the isolates. A high diversity of

Penicillium species was found at low frequencies except forPenicillium citrinum that showed a relative density of 28%(Fig. 4). Table 1 shows the ability of Aspergillus and Peni-cillium species isolated from feedstuffs and finished swinefeed samples to produce OTA when cultured on YESmedium. Seventy-five out of 317 (23.7%) of the total iso-lated strains were able to produce OTA in vitro. The OTAlevels ranged from 9 to 116 mg kg�1. A. ochraceus andP. verrucosum strains produced the highest OTA levels (96.5and 58.2 mg kg�1, respectively), followed by A. carbonariusand A. niger aggregate. Fig. 5 shows the natural OTA levelspresent in all analyzed feedstuffs and finished swine feed.Corn samples (44%) were positive for OTA contaminationwith levels between 42 and 224 mg kg�1. Finished feed(31%) and brewers’ grains samples (25%) were contami-nated with OTA at levels that ranged from 36 to 120 mg kg�1

and 28 to 135 mg kg�1, respectively.

4. Discussion

The mycobiota, OTA-producers strains and natural OTAcontamination from swine feedstuffs were studied. Total

Fig. 3. Relative density (%) of Aspergillus spp. isolated from swine feedstuffs.

Table 1Ochratoxigenic Aspergillus and Penicillium strains and ochratoxinproduction (mg kg�1).

Species Positivestrainsa

OTA-producerspercentage (%)

Rangeb

(mg kg�1)Mean levels� SDc

A. niger 43/175 24.6 10–26 17.2� 5.3A. carbonarius 5/7 71.4 9–32 22.6� 7.3A. ochraceus 19/74 25.6 53–116 96.5� 18.6P. verrucosum 8/61 13.1 18–97 52.8� 11.7

a Number of producer strains vs. total strains.b Minor and major levels of OTA.c Mean level of OTA� standard deviation (SD).

C.A.R. Rosa et al. / Toxicon 53 (2009) 283–288286

mold counts on DRBC from all analyzed substrates excee-ded the levels proposed as hygienic feed quality limits(GMP, 2008). Oliveira et al. (2006) and Fraga et al. (2007)have studied the mycoflora in poultry feed from Brazil andfound values for total mold counts of 103 CFU g�1.However, counts higher than 1�105 CFU g�1 have beenfound in a similar study (Rosa et al., 2006). An analysis ofthe mycobiota of equine feed samples in Brazil alsoshowed similar results (Keller et al., 2007); in contrastSimas et al. (2007) obtained levels lower than 103 CFUs g�1

in barley by-products intended to cattle feeding. InArgentina, Dalcero et al. (1997, 1998) obtained total moldcounts similar to ours in poultry feed, whereas GonzalezPereyra et al. (2008) found total molds counts over1.0�105 CFU g�1 in compound feed and home-corn grainsintended for fattening pigs. High levels of molds in feedcould affect the palatability and reduce the nutrientadsorption (Ogundero, 1987; Martins and Martins, 2001).Optimum conditions of temperature and aW for moldproliferation and mycotoxin production can occur duringstorage. In this study, Fusarium spp., Aspergillus spp. andPenicillium spp. prevailed, including potential mycotoxin-producing species such as A. flavus, A. niger aggregate,A. parasiticus, Aspergillus fumigatus and P. citrinum. A highnumber of yeasts were also isolated from brewers’ grainssamples. These probably were produced during the

Fig. 4. Relative density (%) of Penicillium spp. isolated from swine feedstuffs.

fermentation process and proliferated during the storageof brewers’ grains due to the high water content of thissubstrate. High aW levels could influence germination oflatent conidia on the surface of grains or particles (Samsonet al., 2000). The results obtained here suggest thatmaintaining a low aW is a useful practice to reduce fungaldamage of grains and feedstuffs during storage and thehazard of potential mycotoxin contamination. In ourresearch, a high percentage of potential OTA-producingspecies (23.7%) of Aspergillus and Penicillium genera werefound, and all of them were able to produce OTA in vitro. Inprevious studies, similar results were found in poultry feedsamples from Brazil (46% of OTA producers with toxinlevels between 8 and 120 mg kg�1) (Rosa et al., 2006).However, Fraga et al. (2007) isolated only two OTA-producer strains of A. melleus in this substrate. InArgentina, Magnoli et al. (2002) found results similar toours in several feedstuff materials. A. niger aggregate hasbeen used in food manufacturing, since they were char-acterized with the GRAS (Generally Recognized as Safe)status by the Food and Drug Administration (FDA, USA)(Powell et al., 1994). Our results showed that A. nigeraggregate prevailed in several products, demonstratinga wide dissemination in tropical and subtropical areas.Until recently, the ability of A. niger aggregate and othermembers of Nigri section to produce OTA, was unknown.Accensi et al. (2001) stated that, as A. ochraceus is notwidely disseminated in the tropics, it is possible that theA. niger aggregate strains could be responsible for OTAproduction in tropical and subtropical regions. Our resultsdo not agree with these authors since both species,

Fig. 5. Ochratoxin A (OTA) natural contamination present in swine feedstuffsamples (mg kg�1).

C.A.R. Rosa et al. / Toxicon 53 (2009) 283–288 287

A. ochraceus and A. niger aggregate are widely distributedin our country. Since the first description of OTA produc-tion by A. niger aggregate and A. carbonarius, the signifi-cance of black aspergilli as toxigenic fungi has changed(Abarca et al., 1994; Horie, 1995). These species were fora long time considered as saprophyte environmentalcontaminants and as sometimes opportunistic infectiousagents, but always non-toxigenic (Pitt and Hocking, 1997).Species belonging to Nigri section have a significant role inindustry, being often used in fermentation processes asthey produce different organic acids and hydrolyticenzymes (Teren et al., 1996; Varga et al., 2001). However,recent studies have incriminated the A. niger aggregate asochratoxigenic species contaminant of several substrates(Heenan et al., 1998; Rosa, 2002). Magnoli et al. (2002)demonstrated the absence of OTA in raw materialsbut detected OTA levels in stored feed. Ochratoxin A levelsdetected in the present study were lower than thosedetected by Cruz et al. (1984) in cases of natural ochra-toxicosis in pigs and much lower than the levels reportedby Krogh (1978) for natural porcine nephropatic myco-toxicosis in Denmark. The GMP regulations for animal feedsector established that the action limit for OTA on rationbasis for pigs and piglets is 40 mg kg�1 and the rejectionlimit is 50 mg kg�1 (GMP, 2008). In this study, all samplesexceeded these limits. In a previous study, we found 100%of the analyzed poultry feed samples from Brazil, werecontaminated with OTA levels between 1.3 and 80 mg kg�1

(Rosa et al., 2006). Other studies have shown the effects offeeding pigs feeds contaminated with 0.5–2.5 mg kg�1 OTAincluded reduced feed intake and feed efficiency, reducedweight gain, impaired renal function and nephropathy(Lippold et al., 1992; Glavits, 1993). A high diversity ofmold species was isolated from swine feedstuffs andfinished feed. Several Penicillium spp. isolated such asP. citrinum, P. purpurogenum, Penicillium janczewskii andPenicillium funiculosum are involved in food spoilage andare potential producers of different toxic fungal metabo-lites (citrinin, rubratoxins, griseofulvin, penitrem A, andpatulin). Information regarding the toxicological effects ofthese mycotoxins in animals is scarce (Leung et al., 2006).However, we also found potential fumonisins and tricho-thecenes producers (Fusarium isolates), and A. flavus,A. parasiticus and A. fumigatus, potential producers ofaflatoxins and gliotoxin, respectively. This is the firstscientific report on contamination by ochratoxigenic moldsand OTA in feedstuffs and finished feed intended for swinein Brazil. The presence of OTA in feedstuffs and rationsrequires periodic monitoring in order to prevent theoccurrence of mycotoxicosis in animal production, reduceeconomic losses and minimize hazards to human health.

Acknowledgements

This work was carried out with grants from CAPES-DSand FAPUR/UFRRJ (Brazil), and FONCYT-PICTO, SECYT(UNRC) and CONICET (Argentina).

Conflict of interest

The author declares that there are no conflict of interest.

References

Abarca, M.L., Bragulat, M.R., Castella, G., Cabanes, F.J., 1994. Ochratoxin Aproduction by strains of Aspergillus niger var. niger. Applied andEnvironmental Microbiology 60, 2650–2652.

Abarca, M.L., Accensi, F., Bragulat, M.R., Cabanes, F.J., 2001. Currentimportance of Ochratoxin A-producing Aspergillus spp. Journal ofFood Protection 64, 903–906.

Accensi, F., Abarca, M.L., Cano, J., Figuera, L., Cabanes, F.J., 2001. Distri-bution of ochratoxin A producing strains in the Aspergillus nigeraggregate. Antonie Van Leeuwenhoek 79, 365–370.

Castillo, M.D., Gonzalez, H.H.L., Martınez, E.J., Pacin, A.M., Resnik, S.L.,2004. Mycoflora and potential production of freshly harvested blackbean from the Argentinean main production area. Mycopathologia158, 107–112.

Cruz, L.C.H., Rosa, C.A.R., Campos, J.C., Turatti, J.A., 1984. Ocratoxicose emsuınos no Estado de Santa Catarina. Revista Brasileira Medicina Vet-erinaria 6 (1), 17.

Dalcero, A.M., Magnoli, C., Chiacchiera, S., Palacio, G., Reynoso, M., 1997.Mycoflora and incidence of aflatoxin B1, zearalenone and deoxy-nivalenol in poultry feeds in Argentina. Mycopathologia 137, 179–184.

Dalcero, A., Magnoli, C., Luna, M., Ancasi, G., Reynoso, M.M.,Chiacchiera, S., Miazzo, R., Palacio, G., 1998. Mycoflora and naturallyoccurring mycotoxins in poultry feeds in Argentina. Mycopathologia141, 37–43.

D’Mello, J.P.F., Macdonald, A.M.C., 1998. Mycotoxins. Animal Feed Scienceand Technology 69, 155–166.

Fagundes, M.H., 2003. Sementes de cevada. <http://www.conab.gov.br/download/cas/especiais/CEVADA-SEMENTE.pdf>.

Fraga, M.E., Curvello, F.A., Gatti, M.J., Cavaglieri, L.R., Dalcero, A.M.,Rosa, C.A.R., 2007. Potential aflatoxin and ochratoxin A production byAspergillus species in poultry feed processing. Veterinary ResearchCommunications 31 (3), 343–353.

GMP: Certification Scheme Animal Feed Sector, 2006. Version 2008.Appendix 1: Product Standards (Including Residue Standards). TheHague, The Netherlands: Productschap Diervoeder. pp. 1–39.

Glavits, R., 1993. Nephropathy caused by ochratoxin A in a Hungarianswine stock. Magyar Allatorvosok Lapja 48, 343–349.

Gonzalez Pereyra, M.L., Pereyra, C.M., Ramirez, M.L., Rosa, C.A.R.,Dalcero, A.M., Cavaglieri, L.R., 2008. Determination of mycobiota andmycotoxins in pig feed in central Argentine. Letters in AppliedMicrobiology 46, 555–561.

Heenan, C.N., Shaw, K.J., Pitt, J.I., 1998. Ochratoxin A production byAspergillus carbonarius and A. niger isolates and detection usingcoconut cream agar. Journal of Food Mycology 1 (2), 67–72.

Horie, Y., 1995. Productivity of ochratoxin A of Aspergillus carbonarius inAspergillus section Nigri. Nippon Kingakukai Kaiho 36, 73–76.

International Agency for Research on Cancer (IARC), 1993. Evaluation ofcarcinogenic risks to humans: some naturally occurring substances;food items and constituents, heterocyclic aromatic amines andmycotoxins. In: Monographs on the Evaluation of Carcinogenic Risksto Humans, vol. 56. IARC, Lyon, pp. 489–521.

Karlovsky, P., 1999. Biological detoxification of fungal toxins and its use inplant breeding, feed and food protection. Journal of Natural Toxins 7,1–23.

Keller, K.M., Queiroz, B.D., Keller, L.A.M., Ribeiro, J.M.M., Cavaglieri, L.R.,Gonzalez, P.M.L., Pereyra, C.M., Dalcero, A.M., Rosa, C.A.R., 2007. Themycobiota and toxicity of equine feeds. Veterinary ResearchCommunications 31 (8), 1037–1045.

Klich, M.A., 2002. Identification of Common Aspergillus Species. Cen-traalbureau voor Schimmelcultures, Utrecht, The Netherlands, p 122.

Krogh, P., 1978. Causal associations of mycotoxic nephropathy. ActaPathologica et Microbiologica Scandinavica [A] 269, 1–28.

Leung, M.C.K., Dıaz-Llano, G., Smith, T.K., 2006. Mycotoxins in pet food:a review on worldwide prevalence and preventative strategies.Journal of Agricultural and Food Chemistry 54, 9623–9635.

Lippold, C.C., Sthoters, S.C., Frohlich, A.A., Boila, R.J., Marquardt, R.R., 1992.Effects of periodic feeding of diets containing ochratoxin A on theperformance and clinical chemistry of pigs from 15 to 50 kg bodyweight. Canadian Journal of Animal Science 72, 135–146.

Lopez-Diaz, T.M., Flannigan, B., 1997. Production of patulin and cytocha-lasin E by Aspergillus clavatus during malting of barley and wheat.International Journal of Food Microbiology 35, 129–136.

Magnoli, C., Chiacchiera, S.M., Miazzo, R., Palacio, G., Angeletti, A.,Hallak, C., Dalcero, A., 2002. The mycobiota and toxicity of feedstuffsfrom a production plant in Cordoba, Argentina. Mycotoxin Research18, 7–22.

Magnoli, C., Astoreca, A., Ponsone, L., Combina, M., Palacio, G., da RochaRosa, C.A., Dalcero, A.M., 2004. Survey of mycoflora and ochratoxin A

C.A.R. Rosa et al. / Toxicon 53 (2009) 283–288288

in dried vine fruits from Argentina markets. Letters in AppliedMicrobiology 39, 326–331.

Magnoli, C., Hallak, C., Chiacchiera, S., Dalcero, A., 2006a. Occurrence ofochratoxin A-producing fungi in commercial corn kernels inArgentina. Mycopathologia 161, 53–58.

Magnoli, C., Astoreca, A., Ponsone, L., Chiacchiera, S., Dalcero, A., 2006b.Ochratoxin A and the occurrence of ochratoxin A-producing blackaspergilli in stored peanut seeds from Cordoba, Argentina. Journal ofthe Science of Food and Agriculture 86, 2369–2373.

Martins, H.M., Martins, M.L., 2001. Mycological quality evaluation ofbovine feedstuffs (Portugal: 1996–1999). Revista Portuguesa deCiencias Veterinarias 96, 85–88.

Ogundero, V.W., 1987. Toxigenic fungi and deterioration of Nigerianpoultry feeds. Mycopathologia 100, 75–83.

Oliveira, G.R., Ribeiro, J.M.M., Fraga, M.E., Cavaglieri, L.R., Direito, G.M.,Keller, K.M., Dalcero, A.M., Rosa, C.A.R., 2006. Mycobiota in poultryfeeds and natural occurrence of aflatoxins, fumonisins and zearalenonein the Rio de Janeiro State, Brazil. Mycopathologia 162 (5), 355–362.

Pitt, J.I., Hocking, A.D. (Eds.), 1997. Fungi and Food Spoilage, second ed.Chapman & Hall, Cambridge, p. 593.

Powell, K.A., Renwick, A., Peberdy, J.F. (Eds.), 1994. The Genus Aspergillus:from Taxonomy and Genetics to Industrial Application. Plenum Press,New York, pp. 303–311.

Rosa, C.A.R., 2002. Micobiota toxıgena e ocratoxinas em raçoes destinadasa alimentaçao de aves, bovinos, suınos e sua importancia em saudeanimal. Universidade Federal Rural do Rio de Janeiro. Tese de Dou-torado (Ciencias Veterinarias), Seropedica, Rio de Janeiro, p 147.

Rosa, C.A.R., Ribeiro, J.M.M., Fraga, M.E., Gatti, M., Cavaglieri, L.R.,Magnoli, C.E., Dalcero, A.M., Lopes, C.W.G., 2006. Mycoflora of poultryfeeds and ochratoxin-producing ability of isolated Aspergillus andPenicillium species. Veterinary Microbiology 113, 89–96.

Rosa, C.A.R., Cavaglieri, L.R., Ribeiro, J.M.M., Keller, K.M., Alonso, V.A.,Chiacchiera, S.M., Dalcero, A.M., Lopes, C.W.G., 2008. Mycobiota andnaturally-occurring ochratoxin A in dairy cattle feed from Rio deJaneiro State, Brazil. World Mycotoxin Journal 1 (2), 195–201.

Samson, R.A., Van Reenen-Hoekstra, E.S., Frisvad, J.C., Filtenborg, O., 2000.Introduction to Food and Airborne Fungi, sixth ed. CentraalbureauVoor Schimmelcultures, Institute of the Royal Netherlands Academyof Arts and Sciences, Utrecht, The Netherlands, p. 388.

Scudamore, K.A., MacDonald, S.J., 1998. A collaborative study of an HPLCmethod for determination of ochratoxin A in wheat usinginmunOTAffinity column clean-up. Food Additives and Contaminants15, 401–410.

Shlosberg, A., Elkin, N., Malkinson, M., Orgad, U., Hanji, V., Bogin, E.,Weisman, Y., Meroz, M., Bock, R., 1997. Severe hepatopathy in geeseand broilers associated with ochratoxin in their feed. Mycopathologia138, 71–76.

Simas, M.S., Botura, M.B., Correa, B., Sabino, M., Mallmann, C.A.,Bitencourt, T.C., Batatinha, M.J., 2007. Determination of fungalmicrobiota and mycotoxins in brewers grain used in dairy cattlefeeding in the State of Bahia, Brazil. Food Control 18, 404–408.

Teren, J., Varga, J., Hamari, Z., Rinyu, E., Kevei, F., 1996. Immunochemicaldetection of ochratoxin A in black Aspergillus strains. Mycopathologia134, 171–176.

Van Egmond, H.P., Speijers, G.J.A., 1994. Survey of data on the incidenceand levels of ochratoxin A in food and animal feed worldwide. Journalof Natural Toxins 3, 125–144.

Varga, J., Rigo, K., Teren, J., 2001. Degradation of ochratoxin A by Asper-gillus species. International Journal of Food Microbiology 59, 1–7.

Visconti, A., Bottalico, A., 1983. High levels of ochratoxins A and B inmoldy bread responsible for mycotoxicosis in farm animals. Journal ofAgricultural and Food Chemistry 31, 1122–1123.