Mycotoxins survey in feed materials and feedingstuffs in years 2015-2020.

Mycotoxins are secondary metabolites produced by several fungi, mainly from Penicillium, Fusarium, and Aspregillus genera. They can contaminate many raw materials both in the field and during storage under very different conditions. This study aimed to evaluate mycotoxin contamination levels of raw materials and products for animals in Poland in the years 2015-2020. Producers, farmers, and veterinary doctors delivered a total of 3980 samples (642 maize samples, 2027 feed samples, 990 small grain samples, 142 maize silage samples and 179 TMR samples). Samples were analyzed for the occurrence of several mycotoxins, including aflatoxins, fumonisins, ochratoxin A, deoxynivalenol, nivalenol, T-2 toxin, H-2 toxin, and zearalenone. Trichothecenes, zearalenone, and fumonisins were determined using the LC-MS/MS technique. Ochratoxin A and aflatoxins were determined using the HPLC-FLD method. Deoxynivalenol and zearalenone were the most common contamination in maize samples (97.3% and 98.4%, respectively) and feed samples (99.7% and 100% samples, respectively). They were also present in all maize silage and TMR samples. The highest concentration levels of deoxynivalenol and zearalenone were 16,889 µg/kg in the wheat sample and 1420 µg/kg in the maize sample. Additionally, in 51 samples, mycotoxins level (mainly deoxynivalenol and zearalenone levels) was above recommended levels set by the European Union. The present study showed that both feeding and raw materials are contaminated by mycotoxins, often by more than one.

Virulence markers associated with Trueperella pyogenes infections in livestock and companion animals.

Trueperella pyogenes is an opportunistic pathogen that causes diverse pyogenic infections in livestock. The genes that encode the exotoxin pyolysin (plo) and other putative factors that promote adhesion of pathogen to host cells (fimbriae fimA, fimC, fimE, fimG, neuraminidases nanH, nanP, and collagen-binding protein cbpA) have been associated with virulence, particularly in mastitis and uterus infections of dairy cows. However, the role of these virulence markers in the pathogenicity of the agent in domestic animals infections still is incompletely understood. The genes plo, fimA, fimC, fimE, fimG, nanH, nanP, and cbpA were investigated in 71 T. pyogenes strains recovered from cattle, sheep, goats, dogs, equines, and a pig, recovered from mastitis (n = 35), and non-mastitis (n = 36) cases (abscesses, reproductive tract diseases, pneumonia, lymphadenitis, encephalitis). The most common genes harboured by the isolates were: plo (71/71 = 100·0%), fimA (70/71 = 98·6%), nanP (56/71 = 78·9%), fimE (53/71 = 74·6%), fimC (46/71 = 64·8%) and nanH (45/71 = 63·4%), whereas cbpA (6/71 = 8·4%) and fimG (4/71 = 5·6%) were uncommon. The most frequent genotypes were plo/fimA/fimE/fimC/nanH/nanP (17/71 = 23·9%), plo/fimA/fimE/nanH/nanP (13/71 = 18·3%), and plo/fimA/fimE/fimC/nanP (11/71 = 15·5%). No association was observed between the presence of genes vs clinical signs or host species. To the best of our knowledge, this is the first report on aforementioned virulence factors of pathogen detected in diseased horses and dogs. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of particular virulence factors of Trueperella pyogenes that determine different pyogenic infections among domestic animals is poorly understood. Eight putative virulence genes and genotype profiles of 71 isolates were investigated among different clinical manifestations in domestic animals. The most common genes were plo (71/71 = 100·0%), fimA (70/71 = 98·6%), nanP (56/71 = 78·9%), fimE (53/71 = 74·6%), fimC (46/71 = 64·8%) and nanH (45/71 = 63·4%), whereas plo/fimA/fimE/fimC/nanH/nanP (17/71 = 23·9%), plo/fimA/fimE/nanH/nanP (13/71 = 18·3%), and plo/fimA/fimE/fimC/nanP (11/71 = 15·5%) were the most frequent genotypes. Studies involving virulence factors are critical in the investigation of molecular epidemiology, pathogenicity, and hypothetical differences in the virulence among T. pyogenes strains from different geographical areas.

Determination of mycotoxins, alkaloids, phytochemicals, antioxidants and cytotoxicity in Asiatic ginseng (Ashwagandha, Dong quai, Panax ginseng).

Mycotoxins and selected hazardous alkaloids in the medicinal plants (Panax ginseng, Angelica sinensis, and Withania somnifera) and dietary supplements were determined. Purine alkaloids were found in majority of samples; however, isoquinoline alkaloids were less abundant than indole. The predominant alkaloids appear to be caffeine (purine group), harman (indole group) and berberine (isoquinoline). Examined medicinal plants and dietary supplements were contaminated by mycotoxins (especially ochratoxin A 1.72-5.83 µg kg-1), and many species of mold (e.g. Cladosporium, Eurotium, Aspergillus, Rhizopus, Penicillium). MTT cytotoxicity tests revealed that plant and supplements extracts exhibited medium or high cytotoxicity (only Dong quai-low). Moreover, antioxidant activity, total phenolics content and selected phytochemicals were analyzed by spectrophotometric and chromatographic methods. Quercetin and rutin were predominant flavonols (1.94-9.51 and 2.20-7.28 mg 100 g-1, respectively). Analysis of phenolic acids revealed-gallic acid, as the most abundant, except Panax ginseng, where ferulic acid was prevailing. The results were analyzed by chemometric methods (cluster analysis, ANOVA).

Determination of moulds and mycotoxins in dry dog and cat food using liquid chromatography with mass spectrometry and fluorescence detection.

In this study moulds and 12 mycotoxins in dry pet food samples (25 for dogs and 24 for cats) were determined. Primary moulds identified were Aspergillus, Mucor and Penicillium, found in 55% of the samples. Deoxynivalenol and zearalenone (ZEN) were detected in all samples with mean respective concentrations being 97.3 and 38.3 µg kg(-1) in cat food and 114 and 20.1 µg kg(-1) in dog food. T-2 and HT-2 toxins were present in 88% and 84% of the samples, respectively. Two samples contained fumonisins, with a maximum concentration of 108 µg kg(-1). Aflatoxin B1 and ochratoxin A were detected in 8% and 45% of the samples, respectively. The measured mould and mycotoxin levels were consistent with results obtained by other studies. However, potential exposure to relatively high concentrations of an oestrogen mycotoxin as is ZEN, especially when in combination with other mycotoxins, needs attention.

Deoxynivalenol as a contaminant of broiler feed: effects on bird performance and response to common vaccines.

Recent data suggest that Fusarium trichothecenes may reduce broiler performance at levels previously thought not to affect this variable in chickens. In the present study, we investigated the effects of deoxynivalenol (DON), a type-B trichothecene, on broilers. Male broilers at 7 d of age were fed either a basal diet (0.265 ± 0.048 mg of DON; 0.013 ± 0.001 mg of zearalenone/kg), a low DON diet (1.68 mg of DON/kg; 0.145 ± 0.007 mg of zearalenone/kg), or a high DON diet (12.209 ± 1.149 mg of DON/kg; 1.094 ± 0.244 mg of zearalenone/kg). Increasing levels of DON decreased the weekly weight gain linearly (P ≤ 0.041) during the first 3 wk of exposure; there were no significant differences in the weight gain of the birds after wk 3. With increasing levels of DON, the titers against Newcastle disease virus increased linearly during wk 2 (P = 0.022) and wk 4 (P = 0.033) of exposure, whereas the titers against infectious bronchitis virus decreased linearly (P = 0.006) during wk 5 of exposure. The serum protein concentration increased linearly (P = 0.017) during wk 2 and quadratically (P = 0.002) during wk 4 of exposure. Under these experimental conditions, the performance and vaccine response of the broilers were modulated to varying degrees at concentrations of DON that are currently permitted (up to 5 mg/kg of diet) in many countries. Further studies are therefore required to clarify the implications of these results on the welfare of chickens.

Occurrence of mycotoxins in Polish animal feed in years 2006-2009.

We performed a 4-year survey (2006-2009, 1255 samples) of fungal secondary metabolites in feed material (cereal and corn grains) and feedstuffs (silages, mixed feeds). Five major mycotoxin groups were studied, including aflatoxins (AF), ochratoxin A (OTA), trichothecenes [deoxynivalenol (DON), nivalenol (NIV), T-2 toxin, HT-2 toxin], zearalenone (ZEA) and fumonisins (FUM). The metabolites were identified using HPLC methods with fluorescent, UV and MS/MS detection. Both immunoaffinity and SPE columns were used for sample preparation. In eleven samples, the concentration of several mycotoxins exceeded the recommended guidelines for feedstuffs. DON was detected at the highest concentration in the majority of analysed samples (cereal grains, silages and mixed feeds, maximum values ranged from 409 to 14,470 ng/g). Corn grains also contained other Fusarium toxins (FUM) at maximum levels ranging from 435 to 9409 ng/g. The highest average(positive) concentration of the other trichothecenes (NIV, T-2 and HT-2 toxins) was <5.0-139 ng/g. ZEA was found at the highest concentration in corn grains and silages (maximum values ranging from 292 to 603 ng/g and 116 to 1150 ng/g, respectively). The highest average(positive) concentration and the maximum level of OTA were detected in cereal grains (33.0 ng/g in 2009 and 760 ng/g in 2007, respectively). Less than 7% of the 557 samples were contaminated with AF at low levels (maximum of 0.61 ng/g). Our results support the need for further monitoring of mycotoxins in Polish feedstuffs and their components.

Gross intestinal adaptations in relation to broiler performance during chronic aflatoxin exposure.

The present trial was conducted to study some morphological, digestive, and electrophysiological variables of the small intestine during chronic exposure of broilers to aflatoxin B(1) (AFB(1)). Ross 308 male chicks (7 d old) were randomly allotted to control (no AFB(1)), low AFB(1) (0.07 mg of AFB(1)/kg), or high AFB(1) (0.75 mg of AFB(1)/kg) diet. The high AFB(1) diet resulted in reduced (P ≤ 0.002) bird performance during the first 4 wk of exposure, whereas the low AFB(1) diet temporarily reduced (P = 0.034) the bird performance during wk 3 of exposure. During wk 4 of exposure, a linear (P ≤ 0.013) decrease in the unit weight of both the duodenum and jejunum was observed with increasing levels of AFB(1). This reduction in unit weight appeared to progress from the proximal (duodenum) to the distal (jejunum) small intestine with increase in the length of exposure and was not accompanied by modulation of electrophysiological variables in jejunal epithelium. Response from amiloride, a specific blocker of epithelial sodium channel, was also similar among jejunal epithelia of birds under different treatments. Interestingly, a compensatory linear (P ≤ 0.002) increase in the length of the duodenum and jejunum under high AFB(1) diets was noted to occur during wk 4 of exposure. Thus, retention of DM and nitrogen was not negatively affected by the AFB(1) diets. These data indicate that the intestine in broilers may adapt to an ongoing dietary challenge to AFB(1).

Cytotoxicity of Aspergillus strains isolated from the neonatal intensive care unit environment.

PURPOSE: It is essential in the medical care for immunocompromised patients that the environment should create minimal infection risk. Particularly, it concerns fungal infections. Fungi belonging to the Aspergillus genus are second frequent group of nosocomial pathogens producing opportunistic infections. MATERIAL AND METHODS: The objective of the study was evaluation of prevalence and cytotoxicity of selected Aspergillus strains isolated from the neonatal intensive care unit environment. During 5 consecutive days, 30 samples of indoor air and 120 imprints from the ward environment were collected. Cytotoxicity of 17 strains was evaluated using a MTT (3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide) test. RESULTS: Mean numbers of fungi in the indoor air varied from 50 to 2370 c.f.u.xm-3 while in the imprints from 0.04 to 8.83 c.f.u.xcm-2. Moulds were isolated from most of the materials. All of the 17 strains subject to MTT test were cytotoxic. CONCLUSION: All of the Aspergillus strains isolated from the neonatal intensive care unit were cytotoxic. Cytotoxic Aspergillus strains present in the environment of immunocompromised patients may be potentially dangerous to them.

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.

The level of ochratoxin a in patients after nephrectomy.

Ochratoxin A (OTA) was analyzed in human serum and kidney samples, collected in Poland in the Pomeranian region from March to September 2005. OTA was determined using reversed phase liquid chromatography with fluorescence detection. The collected samples were from patients after nephrectomy (9 from men and 11 from women) and control serum samples from people without kidney diseases (3 and 3, respectively). The mean concentration OTA in serum of the healthy group was 0.37 ng/ml in both men and women. In patients subjected to nephrectomy it reached 1.06 ng/ml in men and 0.94 ng/ml in women, the mean content of ochratoxin A in kidneys was 0.23 ng/g and 0.20 ng/g, respectively. The highest concentration of OTA in serum among the patients subjected to nephrectomy was 3.77 ng/ml in men and 2.27 ng/ml in women while in kidneys 0.45 ng/g and 0.39 ng/g, respectively.

[Not Available]. / Gehalt von Ochratoxin A und Ergosterol in den Proben von Malz und Bier aus einer lokalen Brauerei.

Growing consumption of beer in present diet may lead to higher risk of piling up of ochratoxin A. In our studies we evaluated malt and beer from the local Pomeranian brewery which offers its products at the market in the lowest price. The evaluation had been conducted for 3 months during winter time and the content of fungi, OTA and ergosterol had been analyzed.High concentration of ergosterol was found in all the malt samples, whereas ochratoxin A was present in one delivery of the product only. In contrast, in 70% beer samples low concentration of OTA was found.

[Not Available]. / 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.

The effect of high and low doses of ochratoxin A in feed on growing chicken.

Effects of high and low dose of ochratoxin A (OTA) as pure toxin supplemented to feed were investigated on the performance of growing chicken. Two groups were fed with different doses of chemical pure OTA 0.5 ppm and 5 ppm in feed and the effects of toxin on body and organ weights were studied and compared with control group. No effects were observed by feeding 0.5 ppm OTA, whereas 5 ppm had a negative effect on the daily body mass gain. OTA in feed had a negative effect on the daily body mass gain. In contrary nephrotoxic effects could be observed by feeding the naturally OTA contaminated feed with only 0.2 ppm of OTA produced byPenicillium verrucosum.