Micoflora and aflatoxigenic strains in commercial pet food in Argentina

FERNANDEZ-JURI M. G.,; ASTORECA, A.,; BARBERIS C.,; ASILI R.,; ROSA C.A.R.,; DALCERO A.M.,; MAGNOLI C.

Nantes, Aperdpa Francia

Congreso; 10th Congress of the European Society of Veterinary and Comparative Nutrition, Congress Proceedings.; 2006

INTRODUCTION Information about fungi associated with foods and feeds is important in assessing risk of mycotoxin contamination. Dog and cat food are prepared with vegetables and/or meat, cereal grains, vitamins and minerals. The filamentous moulds most commonly found in stored cereal grains are Aspergillus, Penicillium and Fusarium species. These fungi can cause food spoilage, biodeterioration and are capable of producing different Mycotoxins. Aspergillus species (A. flavus, A. parasiticus, A. nomius) are the more common toxigenic species in various feeds The canine seems to be sensitive to a chronic low level intake of AFB1. These animals exhibited an acute clinical syndrome after chronic consumption of the contaminated food for 90 to 120 days. OBJECTIVES The aims of this work were to isolate and identify the fungal flora and determine aflatoxins (AFs) in dry pet foods, and evaluate the toxigenic capacity by strains of Aspergillus section Flavi. MATERIALS AND METHODS A total of seventeen samples of dry pelleted pet foods were collected from a market located in the south of Córdoba province, Argentina. Isolation of moulds was carried out by the spread surface method. The cultures media used were dichloran-rose-bengal-chloranphenicol agar (DRBC) and dichloran-chloranphenicol 18% glycerol agar (DG18). The average number of colonies on the triplicates plates was recorded and the number of mould refers to CFU per gram of sample. Each strain isolated was identified to genera level (Pitt and Hocking, 1997). Aspergillus species were identified according to Klich (2002). The strains of A. flavus were evaluated for their ability to produce AFs extract malt agar (MEA) according to Geissen (1996), and detection by TLC. The determination of AFs in samples was realized by TLC (AOAC, 2003) and HPLC (Trucksses et al. 1994). For HPLC determination, the extraction of AFs in pet food samples was carried out with acetonitrile- water, cleanup by MycoSep® 228 AflaPat (Romer, Lab). RESULTS Mycological examination of the pet food samples indicated the presence of 13 genera of filamentous fungi (Fig. 1). The most frequent moulds were Aspergillus spp. (29% and 53%), Penicillium spp. (35% and 29%) and Fusarium spp. (23% and 29%), isolated from DRBC and DG18, respectively. The mean of total fungal count was 1.9 x 103 and 6.62 x 102 CFUg-1 on DRBC and DG18 media, respectively. A 40% of A. flavus strains was aflatoxin- producing. The levels of AFB1 produced by these strains ranged from 0.23 to 66.25 µg g-1 (Table 2). None of the samples assayed by TLC showed detectable levels of AFs (AFB1, AFB2, AFG1 and AFG2). The HPLC analysis showed that AFB1 was found in one sample of the pet feed at level of 0.52 ppb, whereas AFB2, AFG1 and AFG2 were not detected. CONCLUSION The total mould count in this substrate was smaller than the limit to assure hygienic sanitary quality (104 CFUg-1). The data presented in this study confirm the aflatoxigenic fungi contamination could represent a hazard for cats and dogs. Careful control of raw material and environmental conditions in dry pet food elaboration are recommended in order to preserve the toxigenic fungi growth and mycotoxins production. REFERENCES Trucksess M.W., Stack M.E., Nesheim S., Albert R., and. Romer T. (1994). Multifunctional column coupled with liquid chromatography for determination of aflatoxins B1, B2, G1 and G2 in corn, almonds. Brazil nuts, peanuts, and pistachio nuts: Collaborative study. J. AOAC Int., 77: 1512-1521. Klich, M.A. (2002). Identification of Common Aspergillus Species, Utrecht, The Netherlands. Geissen, R. (1996). Multiplex polymerase chain reaction for the detection of potential aflatoxin and sterigmatocystin producing fungi. J. Off. Appl. Microbiol. 19, 388-392. Official methods of analysis of AOAC International. (2003). 17th edition. 2nd revision. Gaithersburg, MD, USA, Association of Analytical Communities. Table 1. Total mould count of Aspergillus spp, Penicillium spp., Fusarium spp. and Eurotium spp. in dry pet food from Argentina Total mould count (CFU/g) DRBC DG18 Species Range Mean ± SD Range Mean ± SD A. candidus - - - 1 x 102 ± 0.0 A. carbonarius - 80 ± 0.0 - - A. flavus 1 x 102 - 6 x 102 4 x 102 ± 2.8 x 102 1 x 102 - 6 x 102 4.5 x 102 ± 4.4 x 102 A. foetidus - 1 x 102 ± 0.0 - 1 x 102 ± 0.0 A. fumigatus - 1.5 x 102 ± 70 - 1 x 102 ± 0.0 A. terreus - 2.5 x 102 ± 0.0 - - P. aureantiogriseum - - - 1 x 102 ± 0.0 P. chrysogenum 1 x 102 - 3 x 102 2 x 102 ± 1.41 x 102 - 1 x 102 ± 0.0 P. citrinum - 1 x 102 ± 0.0 - - P. crustosum - - 1 x 102 - 3 x 102 2 x 102 ± 1.4 x 102 P. implicatum - 8.3 x 103 ± 0.0 - 1 x 102 ± 0.0 P. oxalicum - - - 1 x 102 ± 0.0 F. proliferatum - - - 1 x 102 ± 0.0 F: verticillioides - 1.4 x 103 ± 0.0 1 x 102 - 1.45 x103 1.45 x 103 ± 3.5 x 103 E. repens - 1 x 102 ± 0.0 - - E. amstelodami 1 x 103 - 2 x 103 1.5 x 103 ± 7.1 x 102 - - E. chevalieri - - - 1 x 102 ± 0.0 SD: standard deviation. n= 17. Table 2. Aflatoxins production by Aspergillus section Flavi isolated from pet feeds from Argentina. Species Positive strains* Range of AFB1 (µg g-1) Mean of AFB1