Jan Grajewski

Study on biodegradation of some A- and B-trichothecenes and ochratoxin A by use of probiotic microorganisms.

In vitro biodegradation experiments were done using some probiotic microorganisms. DifferentSaccharomyces cerevisiae, Lactobacilli andBacilli strains were tested for their ability to degrade Nivalenol (NIV), Deoxynivalenol (DON), Diacetoxyscirpenol (DAS), T2-Toxin and Ochratoxin A (OTA). The concentrations of selected mycotoxins were in the range of natural occurring toxin contaminations (1ppm for NIV and DON, 500ppb for DAS and T-2 and 50ppb for OTA). No alteration of concentrations could be registered for the tested trichothecenes. The best results could be achieved in experiments with OTA by up to 94% detoxification. Influence of toxins on colony forming unit of all tested microorganisms were recorded. Especially T-2 toxin and DAS have a slowing effect on growth of some strains.

The Impact of the Fusarium Mycotoxin Deoxynivalenol on the Health and Performance of Broiler Chickens

The aim of the present experiment was to investigate the effects of feeding grains naturally contaminated with Fusarium mycotoxins on morphometric indices of jejunum and to follow the passage of deoxynivalenol (DON) through subsequent segments of the digestive tract of broilers. A total of 45 1-d-old broiler chickens (Ross 308 males) were randomly allotted to three dietary treatments (15 birds/treatment): (1) control diet; (2) diet contaminated with 1 mg DON/kg feed; (3) diet contaminated with 5 mg DON/kg feed for five weeks. None of the zootechnical traits (body weight, body weight gain, feed intake, and feed conversion) responded to increased DON levels in the diet. However, DON at both dietary levels (1 mg and 5 mg DON/kg feed) significantly altered the small intestinal morphology. In the jejunum, the villi were significantly (P < 0.01) shorter in both DON treated groups compared with the controls. Furthermore, the dietary inclusion of DON decreased (P < 0.05) the villus surface area in both DON treated groups. The absolute or relative organ weights (liver, heart, proventriculus, gizzard, small intestine, spleen, pancreas, colon, cecum, bursa of Fabricius and thymus) were not altered (P > 0.05) in broilers fed the diet containing DON compared with controls. DON and de-epoxy-DON (DOM-1) were analyzed in serum, bile, liver, feces and digesta from consecutive segments of the digestive tract (gizzard, cecum, and rectum). Concentrations of DON and its metabolite DOM-1 in serum, bile, and liver were lower than the detection limits of the applied liquid chromatography coupled with mass spectrometry (LC-MS/MS) method. Only about 10 to 12% and 6% of the ingested DON was recovered in gizzard and feces, irrespective of the dietary DON-concentration. However, the DON recovery in the cecum as percentage of DON-intake varied between 18 to 22% and was not influenced by dietary DON-concentration. Interestingly, in the present trial, DOM-1 did not appear in the large intestine and in feces. The results indicate that deepoxydation in the present study hardly occurred in the distal segments of the digestive tract, assuming that the complete de-epoxydation occurs in the proximal small intestine where the majority of the parent toxin is absorbed. In conclusion, diets with DON contamination below levels that induce a negative impact on performance could alter small intestinal morphology in broilers. Additionally, the results confirm that the majority of the ingested DON quickly disappears through the gastrointestinal tract.

Detection of 3-nitropropionic acid and cytotoxicity inMucor circinelloides.

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 generaPenicillium, Aspergillus, orFusarium. 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 micro-morphology asAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer. The cytotoxicity ofMucor circinelloides extract was analysed using the cytotoxicity test (MTT assay) and the result, showed a low cytotoxicity. Additionally extracts fromAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer were tested for mycotoxin-production using an LC/MS/MS-based multimycotoxin method. 3-nitropropionic acid was detected in the culture extract ofMucor circinelloides.

Aerobic and anaerobic in vitro testing of feed additives claiming to detoxify deoxynivalenol and zearalenone

Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins produced by fungi of the genus Fusarium which frequently contaminate maize and grain cereals. Mycotoxin-contaminated feed endangers animal health and leads to economic losses in animal production. Several mycotoxin elimination strategies, including the use of commercially available DON and ZEN detoxifying agents, have been developed. However, frequently there is no scientific proof of the efficacy of such adsorbents and degrading products. We therefore tested 20 commercially available products claiming to detoxify DON and/or ZEN either by biodegradation (4 products) or a combination of degradation and adsorption (16 products) under aerobic and anaerobic conditions at approx. pH 7. Under the applied conditions, a complete reduction of DON and consequent formation of the known non-toxic metabolite DOM-1 was exclusively observed in samples taken from the anaerobic degradation experiment of one product. For all other products, incubated under aerobic and anaerobic conditions, a maximum DON reduction of 17% after 72 h of incubation was detected. Aerobic and anaerobic incubation of only one tested product resulted in complete ZEN reduction as well as in the formation of the less-toxic metabolites DHZEN and HZEN. With this product, 68–97% of the toxin was metabolised within 3 h. After 24 h, a ZEN reduction ≥ 60% was obtained with four additional products during aerobic incubation only. Six of the 20 investigated products produced α- and/or β-ZEL, which are metabolites showing similar oestrogenic activity compared to ZEN. Aerobic and anaerobic degradation to unknown metabolites with unidentified toxicity was obtained with 10 and 3 products, respectively. The results of our study demonstrate the importance of in vitro experiments to critically screen agents claiming mycotoxin detoxification. Graphical Abstract

Hepatitis E Virus in Wild Boar in Northwest Poland: Sensitivity of Methods of Detection

In northwest Poland, 163 blood and 53 fecal samples of wild boars were collected in winter 2012/13 and 2013/14. All blood samples were tested for the presence of hepatitis E virus (HEV) ribonucleic acid (RNA) by two reverse transcription-polymerase chain reaction (RT-PCR) based methods and by anti-HEV IgG enzyme-linked immunosorbent assay (ELISA). About 17.2% of blood samples were seropositive. One-step nested RT-PCR turned out to be too insensitive (11.6% were positive). Therefore a two-step nested RT-PCR was applied where 25.8% of the blood samples were tested positive for HEV RNA. About 50.0% of blood samples positive in ELISA were also positive in two-step nested RT-PCR. The prevalence of HEV RNA in feces was 9.4%. Based on the results of blood (ELISA, PCR) and fecal (PCR) tests, the overall prevalence of HEV in wild boars in northwest Poland was 36.8%. There was no correlation between the ELISA results and the presence of HEV RNA in plasma or in feces. According to the sequencing results of 348 bp PCR products of HEV, there were four different subtypes identified. Reports on the prevalence of HEV in wild boar populations are varying due to different sensitivities of the detection methods. However, this study reveals based on a highly sensitive method that HEV is widely spread in wild boar populations in the northwestern region of Poland and posing a potential risk to the consumer of game meat.

Untersuchungen unterschiedlicher Baumaterialien auf das Vorkommen von Schimmelpilzen und deren Mykotoxinen

In 1997, large areas in South Poland were affected by a flood of the River Oder. As a immediate measure to support shelterless people living in these areas, simple pavillons were built, which two years later had to be evacuated because of high contamination with moulds. The rate of mould contamination on the walls in interior microclimate and the occurrence of OTA was estimated in two of these pavillions. The air-flow and the moisture were regulated in the experimental rooms where wallpaper was used as the research material. The gypsum cardboards on the walls were also observed. It was found that the moulds were of different species and the number of them was constantly changing irrespective of the accepted configuration of the research position. In every single wallpaper sample OTA was detected. The greatest amount of OTA was found in the pavillions with humidified air. The test of gipsum cardboard walls in pavilions chosen at random, showed an enormous amount of moulds. However, the OTA was not detected.In 1997, large areas in South Poland were affected by a flood of the River Oder. As a immediate measure to support shelterless people living in these areas, simple pavillons were built, which two years later had to be evacuated because of high contamination with moulds. The rate of mould contamination on the walls in interior microclimate and the occurrence of OTA was estimated in two of these pavillions. The air-flow and the moisture were regulated in the experimental rooms where wallpaper was used as the research material. The gypsum cardboards on the walls were also observed. It was found that the moulds were of different species and the number of them was constantly changing irrespective of the accepted configuration of the research position. In every single wallpaper sample OTA was detected. The greatest amount of OTA was found in the pavillions with humidified air. The test of gipsum cardboard walls in pavilions chosen at random, showed an enormous amount of moulds. However, the OTA was not detected.

Food-Borne Pathogens and Contaminants in Raw Milk – A Review

Abstract The aim of the present review is to highlight the threats to human health posed by consumption of milk and dairy products. The interest in drinking raw milk has been growing in some societies as many people believe it has health benefits. Raw milk is promoted as ‘health food’ despite the fact that it poses a realistic microbiological hazard for the consumers’ health or life. Food-borne disease outbreaks associated with Campylobacter spp., Salmonella spp., shigatoxin-producing Escherichia coli, Brucella melitensis, Mycobacterium bovis and tick-borne encephalitis virus have been traced to the consumption of raw milk, however, many other microorganisms that can be present in milk are considered as potential food-borne pathogens to humans. The other common causative agents in food-borne disease outbreaks are bacterial toxins produced by Bacillus, Clostridium and Staphylococcus spp. Some of the milk pathogens harbour antimicrobial resistant genes, which can be transferred to commensal bacteria. Most dangerous are methicillin-resistant Staphylococcus aureus and extended spectrum beta lactamase/AmpC gene-carrying bacteria from the family of Enterobacteriaceae, which might negatively affect the treatment of infections in humans. Fungi are not considered as food-borne pathogens for humans, however their secondary metabolites, mycotoxins, constitute a potential threat to public health. Mycotoxins or their metabolites detected so far in milk samples include aflatoxins, ochratoxin A, zearalenone and its metabolites, fumonisins, de-epoxy-deoxynivalenol and cyclopiazonic acid.

High levels of ochratoxin A in blood serum and kidneys of wild boars Sus scrofa in Poland

The aim of our study was to evaluate the ochratoxin A (OTA) concentration in the blood serum and kidneys of wild boars Sus scrofa in two consecutive years. We took samples from wild boars hunted in five regions of northwestern Poland during November and December 2006 (N = 39) and throughout 2007 (N = 62). The body weight of the animals ranged from 35 to 100 kg. As a control, we used 20 pigs Sus scrofa domestica of an average body weight of 100 kg. We extracted the OTA and then purified it on immunoaffinity columns. The amount of OTA was determined using HPLC-FLD. The OTA concentration varied among individual animals, some of which had extremely high levels in their blood serum (1,170 ng/ml) and kidneys (97 ng/g). The 2006 average OTA concentration in the serum was similar to the average found in 2007 (6.15 ng/ml and 5.91 ng/ml, respectively). In 2006, the concentration of OTA in the serum of wild boars was > 3 times higher than the concentration found in the serum of pigs. We detected a higher level of OTA in the kidneys of wild boars in both 2006 (1.77 ng/g) and 2007 (2.34 ng/g) than the levels present in the kidneys of pigs (0.59 ng/g). In conclusion, the content of OTA in the serum and kidneys of wild boars changed with year and region. The OTA levels in wild boars from certain regions were much higher than in other regions, and such high levels may cause nephropathy in wild boars and thus pose a possible threat to consumers.

Exposure Assessment to Mycotoxins in a Portuguese Fresh Bread Dough Company by Using a Multi-Biomarker Approach

Mycotoxins are toxic mold metabolites that can persist in environment long after the fungi species responsible for their production disappear. Critical workplace for mycotoxins presence has already been studied and nowadays it is possible to recognize that exposure to mycotoxins through inhalation occurs due to their presence in dust. This study aimed to assess occupational co-exposure to multiple mycotoxins in a fresh bread dough company, an occupational setting not studied until now. Occupational exposure assessment to mycotoxins was done using a LC-MS/MS urinary multi-biomarker approach. Twenty-one workers and nineteen individuals that were used as controls participated in the study. Workers/controls (spot-urine) and environment (settled dust) samples were collected and analyzed. Concerning workers group, DON-GlcA, and OTA were the most prevalent biomarkers (>LOD), 66% and 90.5%, respectively. In the control group, OTA was also one of the most detected (68%) followed by CIT (58%) and DON-GlcA (58%). DON was the mycotoxin measured in high amounts in the settled dust sample (58.2 ng/g). Both workers and controls are exposed to several mycotoxins simultaneously. The workers group, due to their high contact with flour dust, revealed a higher exposure to DON. Considering these results, risk management measures must be applied including specific and adequate health surveillance programs in order to avoid exposure and consequently the associated health consequences.

High domestic pig contribution to the local gene pool of free-living European wild boar: a case study in Poland

Rates of hybridization between wild and domesticated animals appear to be increasing worldwide. Recent results suggest that genetic introgression from domestic swine into European wild boar is much more common in local populations than expected, based on pan-European studies. Thus, we screened the genetic purity of 265 free-living wild boars from two hunting areas in Poland by genotyping the melanocortin receptor 1 gene (MC1R) for polymorphism. Unexpectedly, high numbers of individuals with domestic genes (24%) were identified. This suggests that mixed ancestry may be common in Polish wild boar. Among admixed individuals, backcrosses with domestic pig and/or introgressed wild boars were detected (2%). Multiple commercial domestic pig breeds are possibly involved in the introgression observed in the study populations. In addition, the absence of significant differences in the frequency of wild-type allele among two hunting areas suggests high dispersal of individuals and gene flow among populations. We conclude that further study is needed to better understand the mechanisms and sources of introgression in wild boars in Poland.