World Mycotoxin Journal, February 2009; 2 (1): 85-90

Wageningen Academic P u b l i s h e r s

Aflatoxigenic fungi and aflatoxin B1 in commercial pet food in Brazil

S.G. Campos1, L.M. Keller1,6, L.R. Cavaglieri2,3, C. Krger1, M.G. Fernndez Juri2,4, A.M. Dalcero2,3, C.E. Magnoli2,3 and C.A.R. Rosa1,5
1Departamento

de Microbiologia e Imunologa Veterinria, Universidade Federal Rural do Rio de Janeiro, Instituto de Veterinria, Rio de Janeiro 23890-000, Brazil; 2Departamento de Microbiologa e Inmunologa, Universidad Nacional de Ro Cuarto, Ruta 36 km 601, 5800 Ro Cuarto, Crdoba, Argentina; 3Member of Consejo Nacional de Investigaciones Cientficas y Tecnolgicas, Argentina; 4Fellow of Consejo Nacional de Investigaciones Cientficas y Tcnicas, Argentina; 5Member of Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico, Brazil; 6Fellow of Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico, Brazil; adalcero@exa.unrc.edu.ar Received: 25 January 2008 / Accepted: 11 August 2008 2008 Wageningen Academic Publishers

Abstract
The aims of this study were to determine the aflatoxigenic mycoflora and the incidence of aflatoxin B1 in commercial samples of ready dog food. This in turn demonstrated the ability of the Aspergillus flavus and Aspergillus parasiticus strains to produce aflatoxin B1. 180 samples (standard, premium and super premium) were collected. Aspergillus was the prevalent genera followed by Penicillium and Fusarium. A. flavus and A. parasiticus were the prevalent species. All A. flavus and A. parasiticus strains from super premium samples were able to produce aflatoxin B1, whereas toxigenic strains isolated from standard and premium samples varied from 80 to 100%. A high percentage of ready pet food contaminated by toxigenic species from section Flavi was found and aflatoxin B1 levels were detected. The fungal counts from the three kinds of feed did not exceed the proposed value (1104 cfu/g) and none of the samples exceeded the aflatoxin B1 recommended level (20 ng/g). The presence of A. flavus and A. parasiticus with aflatoxigenic ability could be a potential risk for production of AFB1 in feedstuffs when environmental storage conditions are not adequate.
Keywords: Aspergillus flavus, Aspergillus parasiticus, aflatoxin B1, mycoflora, ready pet food

1. Introduction
Numerous reports of flora on food and feed document the frequent presence of potentially toxigenic fungi. Mycotoxins are secondary metabolites secreted by moulds, mostly belonging to the genera Aspergillus, Penicillium and Fusarium. These moulds are able to produce several toxins that frequently contaminate feeds simultaneously and have synergistic effects among themselves (Yiannikouris and Jouany, 2002). Mycotoxins can occur in a wide variety of agricultural commodities. Due to the diversity of their toxic effects they are considered a risk to consumers of contaminated food.

The occurrence of aflatoxin contamination is demonstrated to be sporadic and highly dependent on environmental conditions. Large populations of Aspergillus flavus and aflatoxin contamination occur each year on different commodities, but serious outbreaks are associated with the interaction of optimum temperatures and rainfall (CAST, 2003; Leung et al., 2006). The mycotoxin incidence and relative importance to animals have not been clearly established. Aflatoxin B1 (AFB1) is the most toxic of the aflatoxins and is a potent liver carcinogen. Substantial evidence also exists that lowlevel exposure to aflatoxin may suppress the immune system and increase susceptibility to disease (Sahoo et al., 2001). In dogs, despite numerous studies, aflatoxicosis continues 85

ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2008.1020

S.G. Campos et al.

to be a problem (Bohm and Razzai-Fazeli, 2005; Rumbeiha, 2002). In previous work, the incidence of several toxigenic species and their mycotoxins in poultry, pig and rabbit feeds was studied (Dalcero et al., 1997, 1998, 2002; Magnoli et al., 1998, 1999, 2002, 2005). From these results, the presence of toxigenic fungi and their important mycotoxins such as aflatoxins, fumonisins, ochratoxin A, was detected. Campos et al. (2008) reported the incidence of mycoflora and aflatoxins in raw materials intended to elaborate ready dog food. Both a high percentage of Aspergillus section Flavi and aflatoxin producers were found. However, they did not compare the toxigenic mycoflora and mycotoxins between different commercial ready dog food qualities. Data from dog feed in this region would be of worldwide interest. The aim of this study was to determine the incidence of AFB1 and the aflatoxigenic mycoflora in commercial samples of ready dog food. The ability of A. flavus/A. parasiticus to produce AFB1 was also quantified.

Before calibrating, Aqua Lab was located in an area with a stable ambient temperature and the reference humidity standards were at ambient temperature. New sample cups for reference humidity standards were used. Sodium chloride (NaCl), which has 0.760 aw, was used as a humidity standard and its aw was compared with the table in the operators manual. When readings of the humidity standard were within the stated ranges, duplicate readings of distilled water were made.

Aflatoxin B1 analyses
Aflatoxin B1 analyses were performed by HPLC according to the methodology proposed by Trucksess et al. (1994). A 50 g portion of each feed was extracted with 100 ml acetonitrile:water (90:10, v/v) during 2 min into a blend jar. The mixture was filtered through filter paper Whatman No. 4 (Whatman, Inc., Clifton, NJ, USA) and a 3 ml aliquot taken and placed into 10 ml culture tube. A multifunctional column (Mycosep 224 MFC, Romer Labs, USA) was slowly pushed into the culture tube. The extract was forced through frit, through a 1-way valve and through packing material. Purified extract (0.5 ml) was collected in column reservoir. An aliquot (200 l) was derivatised with 700 l trifluoroacetic acid-acetic acid-water (20:10:70, v/v). The aflatoxin derivative was analysed by using an HPLC system. Chromatographic separations were performed on a reversed phase column (Silica Gel. VARIAN, 1504.6 mm id., 5 m particle size). Water-methanol-acetonitrile (4:1:1, v/v) was used as mobile phase at 1 ml/min flow rate. Fluorescence of aflatoxin derivatives was recorded at excitation and emission wavelengths of 360 nm and 460 nm, respectively. Standard curves were constructed with different levels of AFB1. The concentration of this toxin was quantified by correlating peak heights of sample extracts with those of standard curves. The detection limit of the analytical method was 0.1 ng/g. Recovery experiments were performed in triplet by spiking AFB1 at levels of 0.5, 2.0, 4.0 and 8.0 g/g in four samples of each kind of ready dog food.

2. Materials and methods

Source of samples
One hundred and eighty packages of commercial ready dog food (standard, premium and super premium) from two factories located in Rio de Janeiro State, Brazil were sampled. From each kind, three samples (10 kg each) destined for puppies and 3 for adults were collected monthly, at random, from March to December 2005. These primary samples were finely ground in a laboratory mill that was disinfected between each sample to ensure that cross-contamination could not occur. They were taken and stored at 4 C for 7 days for fungal and AFB1 analysis. The standard, premium and super premium samples varied in gross protein levels. The composition was taken from the dog food company that provided the samples and is shown in Table 1.

Moisture content of samples

Water activity (aw) determinations of samples were carried out with AQUALAB CX2 (Decagon, Devices, Inc. USA) and calibration was performed before sample measurements. Table 1. Nutritional composition from different kinds of ready dog foods.
Composition (%) Standard Premium Super premium 27.24.5 14.32.3 3.30.9 7.31.4 1.40.3 0.80.2

Mycoflora determination
Quantitative enumeration of fungal propagules was done on a solid medium using the surface spread method by blending 10 g portion of each sample with 90 ml of 0.1% peptone water solution for 30 min. Serial dilutions 10-2 until 10-4 concentrations were made and 0.1 ml aliquots were inoculated in triplicates onto three media. Dichloran rose bengal chloramphenicol agar (DRBC) was used for general fungal enumeration, Dichloran 18% glycerol agar (DG18) for xerophilic fungi (Pitt and Hocking, 1997) and NashSnyder medium for isolation of Fusarium species (Nelson et al., 1983). DRBC and DG18 plates were incubated in darkness at 25 C for 7-10 days. The Nash-Snyder plates World Mycotoxin Journal 2 (1)

Proteins Total fats Gross fibre Total minerals Calcium Phosphorous

18.31.4 5.20.6 6.30.9 10.32.1 1.90.3 0.70.2

20.91.5 6.51.2 5.31.1 9.52.2 1.90.5 0.90.2

86

Aflatoxigenic fungi and aflatoxin B1 in commercial pet food G60 Merck), and using chloroform: acetone (9:1, v/v) as developing solvent. AFB1 concentrations were determined by visual comparison with standards. Chromatograms were dried and observed under 365 nm UV light.

were incubated at 24 C for 7 days, in 12/12 h photoperiod under cold white and black fluorescent lamps. Only plates containing 10-100 colony-forming units (cfu) were used for counting and the results were expressed as cfu/g of sample. On the last day of incubation, individual cfu/g counts for each colony type, considered to be different, were recorded. Taxonomic identification of the different genera was made according to microscopic criteria in accordance with appropriate keys (Pitt and Hocking, 1997; Samson et al., 2002). The different colonies of Aspergillus strains were transferred on malt extract agar (MEA) for later identification of species. The result was expressed as frequency (percentage of samples in which each genus was present) and relative density (percentage of isolation of each species among strains of the same genera) (Castillo et al., 2004).

3. Results
Table 2 shows the AFB1 levels obtained from ready dog foods. Standard samples were the most contaminated with AFB1. All kinds of tested samples had levels lower than 9.43 ng/g and 90% of them did not reach 4.7 ng/g. Table 3 shows the fungal cfu/g and aW levels in different culture media. Fungal counts in DRBC and DG18 media ranged from not detected (nd) to 103 cfu/g. Nash-Snyder medium contained counts lower than the detection limit of the technique (102 cfu/g). Water activity values of all ready dog food samples varied from 0.765 to 0.985. Mycological examination of samples indicated the presence of four genera of filamentous fungi (Table 4). All kinds of samples (standard, premium and super premium) showed similar results. The main genus isolated was Aspergillus followed by Penicillium and Fusarium species belonging to Cladosporium and Mucorales were isolated at lower frequencies. Figure 1 shows the relative density (%) of Aspergillus species isolated from standard, premium and super premium ready dog food. Samples showed a high diversity of Aspergillus species. Seven Aspergillus spp. were isolated. A. flavus and A. parasiticus (both belonging to Aspergillus section Flavi) were the most prevalent of all the different samples tested. They were followed by A. niger and A. ochraceus (lower than 15%), A. fumigatus, A. candidus and A. flavipes (lower than 8%). One-hundred percent of A. flavus and A. parasiticus strains isolated from super premium samples were able to produce AFB1, whereas toxigenic strains isolated from standard and premium samples varied from 80 to 100% (data not shown).

Identification of Aspergillus species

For Aspergillus identification, cultures were grown on Czapek yeast extract agar (CYA), at 25 and 37 C; MEA, 25% glycerol nitrate agar (G25N), and Czapek yeast extract with 20% sucrose agar (CY20S), at 25 C. All plates were incubated for 7 days. Each strain was identified following the methods proposed by Klich (2002), Pitt and Hocking (1997), and Samson et al. (2000).

Aflatoxin B1 production by A. flavus and A. parasiticus

All A. flavus and A. parasiticus strains isolated from ready dog food were evaluated for their ability to produce AFB1. It was determined following the methodology described by Geisen (1996). The strains were grown at 25 C for 7 days in MEA in darkness, after which mycelium and conidia were collected from the agar surface with a sterile brush and transferred to Eppendorf tube. Aflatoxin B1 was extracted with 500 l chloroform and centrifuged at 4,000 g for 10 min. The chloroform phase was transferred to a clean tube, evaporated to dryness and re-dissolved in chloroform. The extract was spotted together with standards and screened for AFB1 by thin layer chromatography (TLC) method on silica gel plates without a fluorescent indicator (0.25 mm,

Table 2. Levels of aflatoxin B1 from different kinds of ready dog food.

Ready dog food Contamination frequency (%) AFB1 levels (ng/g)1 Range Standard Premium Super premium
1 AFB 1

90th percentile rank Mean 7.6 7.0 6.0 4.74 3.11 2.68

94.4 57.1 60.0

>0.3-9.43 >0.3-8.11 >0.3-6.38

was detected by HPLC; limit of detection is 0.1 ppb.

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Table 3. Total fungal counts from each kind of ready dog food isolated in different culture media.
Samples Water activity (range) Culture media1 (Log 10) DRBC Range2 Standard Premium Super premium
1 Maximum 2 nd=

DG18 Mean 3.3 <2 <2 Range2 2.2-3.6 2.3-2.6 nd-2.6 Mean 3.3 2.5 <2

NASH2

0.852-0.985 0.805-0.955 0.765-0.874

2.2-3.5 nd-2 nd-2.6

nd nd nd

and minimum values in each culture media. not detected. Detection limit of the technique is 102 cfu/g.

Table 4. Frequency of fungal genera from ready dog food.

Fungal genera Aspergillus spp. Penicillium spp. Fusarium spp. Cladosporium spp. Mucorales Frequency (%) 59.8 20.5 13.7 4.3 1.7

toxin was detected at low concentrations probably because other environmental factors than aW must interact with Aspergillus section Flavi species in order to control the AFB1 production. However, a high level of strains able to produce aflatoxins was isolated from ready dog food. None of the samples exceeded the recommended level AFB1 natural contamination (20 ng/g; GMP, 2005). This toxin has been previously reported as naturally occurring in feed and feedstuffs in Latin America (Dalcero et al., 1997, 1998; Fraga et al., 2007; Keller et al., 2007; Magnoli et al., 1998, 2002, 2005, 2006; Oliveira et al., 2006; Rosa et al., 2006). In this study, although there was a high incidence of A. flavus and A. parasiticus toxigenic species, the second species was the most prevalent. It is necessary to emphasise that A. parasiticus species are more stable as regards the production of aflatoxin and probably more resistant to manufacturing practices for raw materials and ready feeds (Horn, 2007; Pitt and Hocking, 1997). Scudamore et al. (1997) found a high incidence of mycotoxins in raw material for feeds in the UK. In a low number of samples these authors detected AFB1 levels similar to the ones in our study. In contrast, Sharma and Marquez (2001) found 88% contaminated pet food samples with high aflatoxin levels, some of them with levels of 59 and 72 ng/g. Maia and Pereira Bastos de Sequeira (2002) indicated that all pet food samples containing peanut as raw material were positive for AFB1. The occurrence of Aspergillus section Flavi strains and their ability to produce aflatoxins has only been reported by Campos et al. (2008) in Brazil. Aflatoxigenic strain percentages found in this work were higher than those found by Magnoli et al. (1998) and Fraga et al. (2007) in other feedstuffs (ready poultry feed). It has been demonstrated that fungal propagules are helpful indicators for determining feed hygienic quality; these counts should not exceed the values of 104 cfu/g (GMP, 2005). In this work, the fungal counts from the three kinds World Mycotoxin Journal 2 (1)

70 60 Relative density (%) 50 40 30 20 10 0

us tus s s s idu nd ca A. vu nig itic eu iga fla ra s ac fum A. A. oc hr fla vip es er

Standard Premium Super premium

pa

A.

Figure 1. Relative density (%) of Aspergillus species isolated from different kinds of ready pet food.

4. Discussion
Natural occurrence of AFB1 and aflatoxigenic fungi in commercial ready pet food was evaluated. Although aW varied to a large extent, a high percentage of standard, premium and super premium dog food samples was naturally contaminated with low levels of AFB 1. This 88

A.

A.

A.

Aflatoxigenic fungi and aflatoxin B1 in commercial pet food

of feed were not over the proposed value. This result is consistent with that of Martins et al. (2003) who found cfu levels lower than 102 in commercial pet food. Other researchers found counts lower than 104 in cereals and ready feedstuffs (Accensi et al., 2004; Dalcero et al., 1997; Magnoli et al., 2005, 2006). Ready pet foods undergo a pelleting process during their production that decreases the viable fungal propagules. All collected samples were not contaminated with colony forming units per gram over 104. This information corresponds to previous studies that reported a significant decrease of fungal counts when the pelleting process was performed (Chelkowsky, 1991; Dalcero et al., 2002). In this study, Aspergillus section Flavi species (A. flavus and A. parasiticus) were isolated in high frequency and were predominant. This result is not consistent with Bueno et al. (2004) who found only A. flavus as the most prevalent species in Aspergillus genus. Many researchers have proved that most feeds have species from Aspergillus genus as predominant flora. Dalcero et al. (1998) found similar results in mixed feed for rabbit, pig and poultry. In contrast, Scudamore et al. (1997) found Penicillium spp. and Eurotium spp. in pet food for domestic pets and wild birds. Meireles et al. (1994) found Fusarium spp. as the predominant genera, followed by Aspergillus spp. and Penicillium spp. in equine feed. Although very low intake levels of mycotoxins do not cause mycotoxicosis, they do lead to immunity impairment and acquired resistance to infections causing health problems (CAST, 2003; Leung et al., 2006). Specific mycotoxin regulations on pet food should, therefore, be in force to solve the problem. Our results are the first data on natural occurrence of Aspergillus section Flavi species and AFB1 contamination in different kinds of ready pet food in Rio de Janeiro, Brazil. The presence of A. flavus and A. parasiticus with aflatoxigenic ability poses a potential risk for the production of AFB1 in feedstuffs when environmental storage conditions are not adequate.

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Acknowledgements
This work was carried out with grants from CAPES-D, DUMILHO SA Industria e Comercio, Espirito Santo- Brasil, FAPUR/UFRRJ (Brazil), and SECYT (UNRC), CONICET and FONCYT-PICTO (Argentine). We thank Prof. Susan Vilor for the English version.

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