A survey of occurrence of toxogenic fungi and mycotoxins in pig feed samples - use in evaluation of risk assessment.

In order to assess of risk assessment, the aim of this paper was to provide good and detailed insight into the level of contamination of complete feedmixes intended for fattening swine from mycotoxin-producing fungi and mycotoxins (n=18). Isolation and quantitative enumeration of fungal propagules were done on solid media using the standard microbiological procedure. These plates were incubated the number of colonies was determined and thent on the basis of characteristic colonies and microscopic analysis was performed to identify genera and species of moulds. Isolates identified as Aspergillus and Penicillium species were subjected to molecular characterization of the presence of genes responsible for the synthesis of OTA (polyketide synthase gene-PKS). Total fungal counts (CFU/g) ranged from 0,5x105 do 4x106. From a total samples analysed, seven samples had fungal counts higher than the limit established by Serbian regulations (3x105). During a mycological analysis of complete feedmixes intended for fattening swine, a total of six genera and 14 species of moulds were identified of which the most frequent one was of the genus Penicillium (94,4%) while the moulds from Fusarium genere isolated in 55,5% and Paecilomyces in 44,4% of the samples from investigated localities. Other fungi from the genera Aspergillus (22%), Mycor (11,1%) and Alternaria (5,5%) were represented in a less amount. Polymerase chain reaction (PCR) is a set of 18 isolates of the DNA belonging to families Penicillium and Aspergillus. The sequences of PCR reaction products in three samples were compared with nucleotide sequences of genes for poliketid synthase (PKS) from Penicillium species and found that the samples possess PKS sequence. The traditional methods for identification of ochratoxin-producing fungi are time-consuming and labor-intensive. Rapid and specific detection of ochratoxinproducing fungi is important for ensuring microbiological quality and safety of feed and food.


Introduction
contaminate the whole food chain, from the agricultural cultures to the plate of consumers.Mycotoxins occur sporadically both seasonally and geographically.In The contamination of agricultural commodities farm animals, mycotoxins exert their effects through with fungi able to produce toxic metabolites is one of four primary mechanisms: (1) intake reduction or feed the main worldwide concerns.Discoloration, quality refusal, (2) alteration in nutrient content of feed in deterioration, reduction in commercial value and nutrient absorption and metabolism, (3) effects on the mycotoxin production has been linked to mouldy endocrine and exocrine systems and (4) suppression contaminated foods and feeds.It not only generates of the immune system.In routine animal feed screening, great economic losses [1], but also represents a threat mycotoxins are usually found at relatively low levels.to human and animal health, particularly through the Limited information exists regarding the effects of synthesis of mycotoxins.low levels of multiple mycotoxins in livestock.It has Mycotoxins are naturally occurring secondary been suggested that combinations of mycotoxins at metabolites of several toxigenic microfungi that low concentrations may have negative effects on samples.Isolation and quantitative enumeration of livestock, even though the concentrations of individual fungal propagules were done on solid media using the mycotoxins are well below concentrations reported to surface-spread method by blending a 10 g portion of cause negative effects [2].
each sample with 90ml of 0.1% peptone water solution.The main mycotoxins classes of concern Serial dilutions, 10-1 to 10-6 concentration, were made produced by fungi in the genera Aspergillus, from each material and 0.1ml aliquots were inoculated Penicillium and Fusarium include the aflatoxins, in triplicate on two media potato dextrose agar (PDA) ochratoxin A, trichothecenes and fumonisins.Moisture and Czapek yeast extract agar (CYA) of fungal and/or temperatures is the single most important factor enumeration.After 3-7 days, growing fungal colonies in determining if and how rapidly molds will grow in were transferred to Czapek yeast extract agar (CYA) 0 feeds.Improper storage accompanied by too high a and incubated at 25 C in the dark for 7 days.temperature and elevated moisture content in the grain Macrofungi and moulds were identified to favours further mycotoxin production and leads to genera/species by their macro-and micromorphology reduction in grain quality [3,4].Therefore, rapid and features using appropriate identification keys [8][9][10][11][12].specific detection of mycotoxogenic moulds is 2. Mould identification by molecular methods important for ensuring both microbiological quality and A molecular method was used in order to safety of both feed and food.The currently employed eliminate any uncertainty in mould identification based methods for identification of foodborne molds require on the aforementioned traditional methods.Eighteen culture isolation and application of morphological and isolates were tested using both methods and were physiological tests [5,6], which are time-consuming, subsequently compared to determine accuracy.labor-intensive, and often require mycological

Primer selection expertise [7]. Further, plate count techniques do not
In this work, we used two sets of specific primers, detect dead fungi, which could indicate past AoLC35-12L/AoLC35-12R and AoOTAL/AoOTAR, contamination of a product [5].Nucleic acid-based from a 3.4 kb DNA sequence of a polyketide synthase methods, such as the polymerase chain reaction (PKS) gene from A. ochraceus NRRL 3174.These (PCR) and DNA probes, provide powerful and rapid primer sets were tested by PCR method on different tools for the detection of microorganisms.Although genera of OTA and other mycotoxin producing fungi [13].PCR has been widely used for the detection of 2.2.Fungal strains and culture conditions foodborne bacteria and viruses, its application for Fungal strains identigfied as ochratoxogenic 0 specific detection of foodborne molds is relatively were grown at 25 C on potato dextrose agar (PDA) limited.
(Difco, Fisher Labosi) during 7 days.Then spores were The aims of this work were: 1) to determine the collected with a sterile solution of 0.1% (v/v) Tween 80 0 mycobiota in pig feed samples, 2) to develop and (Fisher Labosi) and stored at -20 C in 25% (v/v) of optimize a PCR for rapid and specific detection of glycerol (Fisher Labosi) before use.Conidia (about 6 ochratoxogenic moulds, 3) to evaluate the feedstuffs' 10 /ml) were inoculated into 250-ml Erlenmeyer flasks mycotoxins contamination, especially aflatoxins, containing 100 ml of potato dextrose broth (PDB) 0 ochratoxin, deoxinivlenol and zearalenone, 3) Also, the (Difco, Fisher Labosi), at 25 C, without shaking for 3 days.The mycelium was harvested by filtration through objectives of this study were to determine the effect of a 0.45 Am filter (Millipore) frozen in liquid nitrogen and the moisture content and water activity (aw), on the 0 then stored at -80 C before nucleic acid extraction.mycobiota presence versus specific mycotoxins contamination.The results obtained from the mycoflora analysis which amplified a 340 bp fragment on genomic DNA.

Genomic
in the samples are presented in Figure 1 and 2.

Detection of PCR product
Occurrence of mycotoxins contamination and mean Gel electrophoresis was used to detect the contamination levels of mycotoxins (mg/kg) in samples presence of the amplified PCR product from each mold originating from region where samples were collected isolate.A volume of 20 Al of PCR products was are presented in Figure 4 and 5. subjected to electrophoresis on a 1.2% agarose gel; 5 5 Total fungal counts ranged from 10 to 40x10 the gel was stained with ethidium bromide, and viewed 5 cfu/g.In our study, the highest fungal count (40x10 under ultraviolet light to detect the presence and size of cfu/g) and average total fungal counts was detected in the amplified DNA product.
pig feed samples collected from region Bogatic.From 2.5 DNA sequencing the 18 samples analysed, seven samples had cfu/g PCR products were purified to remove excess higher than the limit established by Serbian regulations primer using micro concentrators, and then directly 5 (3x10 ) [19] Fungal count is an indicator of the quality of sequenced with the Dye-Deoxy Terminator Cycle 5 feeds and should not exceed 1x10 cfu/g [20].It is worth Sequencing kit in an automated DNA Sequencer (BMR mentioning that in such a samples only Penicillium Genomics-Servizio Sequenziamento, Italy).
species mostly belonging to F. equiseti were isolated.

Chemical analysis
Species deter mination revealed great All pig feed samples were analysed with validated heterogeneity.A total of six genera and 14 species of methods and under quality assurance conditions.moulds were identified.With the exception of Alternaria Moisture content alternata, and Mucor racemosus which occurred only The moisture content of the pig feed samples was in one to two samples, the rest of the species were determined by drying at 105°C until the weight did not found in more than one sample in all locations change further [14].The aw value of the grain was surveyed.The most frequently isolated fungus was measured in a hygroscope (GBX Scientific Instruments differences were found between the median DON and and accurate decision support systems for effective OTA contents for all feed items (p>0.05).Additionally, conservation of grain post-harvest.Improved positive and significant correlations between the level screening techniques are needed for monitoring of contamination of samples by total fungal counts and moulud and mycotoxin occurrence, diagnosing the OTA content (r=0.584) were found, as well as the toxicities and prevention and treatment.The PCR could moisture content and the OTA content (r=0.468).In potentially be used as a rapid tool for screening both feed respect to correlations between the level of and food for the presence of ochratoxogenic strains.. contamination of samples by mycotoxins, between research is the development of management Development of a fungal-specific PCR assay for strategies to reduce the incidence of ochratoxogenic detecting low-level fungi in an indoor environment.Mol.strains, in feedmixes.Serbian producerss will use Cell.Probes 14,[339][340][341][342][343][344][345][346][347][348] these strategies to minimise the risk of mycotoxins A Survey of occurrence of toxogenic fungi and mycotoxins in pig feed samples DNA extraction 200 mg of lyophilized mycelium wasMaterial and methodshomogenized in 800 Al of lysis buffer (100 mM Tris-HCl Samples: The research materials consisted of 18 pH 7.4 (Sigma Aldrich), 20 mM EDTA (Sigma Aldrich), representative pig feed samples which were collected 250 mM NaCl (Sigma Aldrich), 2% w/v SDS (Sigma directly at animal farms from different provinces of Aldrich)) by using a Ultra-Turax (Labo moderne) and 0 Serbia during six month period.The samples (each incubated at 37 C for 30 min with 10 Al of 25 mg/ml about 1 kg) were stored at 4 °C and analysed the day RNase solution (Promega), then added with 10 Al of after collection.On the same day, the moisture content proteinase K (20 mg/ml, Euromedex) were added and 0 of the feeds samples was determined by drying.the mixture was incubated at 65 C for 30 min.A volume Reagents: Standards of AFT, OTA, ZEA and DON of phenol-chloroform-isoamylic alcohol (v/v/v: 25/24/1) were purchased from Sigma-Aldrich Chemie GmbH.(Sigma Aldrich) was added, and the mixture was All other solvents and reagents were analytical grade.vigorouslyvortexed for 5 min.The aqueous phase 1. Isolation and identification of fungal collected after centrifugation (15,000 x g, 15 min) was Culturable fungal spore concentrations are extracted by an equal volume of chloroform (Prolabo).0 presented in terms of colony-forming units (CFU)/g of Genomic DNA was precipitated at -20 C in 2 h with two volumes of 100% ethanol (Prolabo).The DNA was FA-St/1, tastatura model MX 3700/ML 4700) at a pelleted by centrifuging at 15,000xg and washed with 1 temperature of 20°C.ml of 75% ethanol then dried at the room temperature.Analysis for aflatoxins (AFT), ochratoxin A (OTA) 100 Al of water was used to re-suspend genomic DNA.deoxynivalenol (DON) and zearalenone (ZEN).The detection of these mycotoxins in all samples were 2.4.PCR reaction performed by TLC using appropriate methodology [15-PCR was performed with the Taq recombinant 18].polymerase (Invitrogen, USA).Amplification was Statistical analysis carried out in 50 Al reaction mixture containing: 5 Al of Taq polymerase buffer10X, 1.5 Al of 50 mM MgCl2, 1 Al Differences in the mean levels of mycotoxins of dNTP 10 mM of each (Promega), 1 AM of each contamination across the three groups of positive primer, 1.5 U of Taq, about 50 ng of DNA genomic, H2O samples was calculated by analysis of variance and 0 up to 50 Al.Reaction conditions were: 94 C for 4 min then by a Student's t-test.Additional posttests were 0 0 0 applied to evaluate differences between groups with (94 C for 40 s, 58 C for 40 s and 72 C for 40 s) x 35 0 statistically significant variation among means.The cycles followed by an incubation at 72 C for 10 min.The differences with p values smaller than 0.05 were amplified products were examined by agarose gel considered statistically significant.electrophoresis.The b-tubulin gene was used as positive control and primer sequences were: Results and Discussion TubF: ctcgagcgtatgaacgtctac; Mycoflora analysis TubR: aaaccctggaggcagtcgc,

AFugure 1 .
Survey of occurrence of toxogenic fungi and mycotoxins in pig feed samples Total fungal counts (CFU/g) (A) and mean contamination levels of moulds and moisture content (B) ., et.al.(1996):Detection of aflatoxigenic development in grain and feedmixes.Combined data molds in grains by PCR.Appl.Environ.Microbiol.26 (9): will enable us to realize the goal of developing realistic 3270-3273.