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Ecosystem services are an important aspect of grasslands utilization; however, they are often contradictory to their main purpose, which is a production of good quality and safe feed. In this study, we evaluated the difference between grass monocultures and species-rich mixtures in terms of epiphytic microflora and mycotoxin contamination levels. We hypothesized that higher species diversity would lead to higher microbial counts, which could lead to higher mycotoxin contamination risk. Differences in epiphytic fungal, yeast and total amount of microorganisms (CFU g -1) depending on the species diversity in the field has been evaluated by cultivation method. Concentration of deoxynivalenol (DON), zearalenone (ZEN) and aflatoxin B1 (AFB1) was measured by ELISA. Results are suggesting that higher total amount of microorganisms were found in monocultures, however, fungal and yeast counts were higher in species-rich mixtures. Higher species diversity of grasses was related to higher total microbial count (TMC) and yeast colonization of phyllosphere. Our results suggest higher risk of fungal phyllosphere colonization of species-rich mixtures with higher biodiversity and therefore higher risk of mycotoxin contamination of such feed.

Novel use of superabsorbent polymers to deliver copper-based foliar fertilization was tested as a means against fungal mycotoxin production and monitor its effect on nutritional characteristics of wheat. Experiment was located in Žabčice, South Moravia region of the Czech Republic, tested wheat variety was 'Julie'. Differences in fertilization medium (water or superabsorbent polymers) and copper treatments (control, CuO, CuSO4, Cu-EDTA, CuO-nano) were tested. Effect of different variants on percentage of ash, crude protein, crude fat and crude fiber, acid detergent fiber, ash-free neutral detergent fiber, lignin, cellulose and starch were determined. Moreover, concentration of deoxynivalenol and T-2 toxin depending on the variant was measured. The highest amounts of crude fat and crude fiber were observed in variant of Cu-nano with superabsorbent polymers, the lowest in CuO respectively. Higher amount of cellulose was found in SAP control, lowest in water control. There were no significant differences in other nutritional parameters. Of the evaluated mycotoxins, we did not detect the deoxynivalenol in any of our samples. For the second evaluated mycotoxin, there was also no difference observed in T-2 toxin production.

With the rising interest in digestate use as a fertilizer on permanent cultures, there is a need to examine its effects on food and feed quality. This study is focused on the use of digestate in grassland fertilization and its effects on nutritive value parameters such as mycotoxin contamination (deoxynivalenol, aflatoxin, and T-2 toxin) and nutrient content (crude protein, crude fat, crude fiber, ash, nitrogen-free extract, digestibility of organic matter, acid detergent fiber, and ash-free neutral detergent fiber). The experiment was carried out in the Czech Republic, and the effects of fertilization regime, year, and harvest date (summer and fall cuts) on nutritive value were observed. An effect of the year on DON, AFB1, and T-2 contamination levels was observed. An effect of the harvest or fertilization regime on mycotoxin contamination was not observed. Significant differences were observed in the content of all nutrients, except ash, depending on the year. Differences were found only in the case of ADF levels, depending on the harvest date, as well; however, no differences were found between fertilization regimes. Our findings suggest that digestate does not negatively affect fodder in terms of nutritive value nor safety.

The aim of the project was to evaluate the potential of microbial threat to feed safety in the year 2018. Analyses of the epiphytic community of several forage species (clovers, cocksfoot, fescue, festulolium, perennial ryegrass, timothy and trefoil) in variants of fertilized and non-fertilized vegetation were performed. The hypothesis is based on the fact that microorganisms are normally present on plant material during its growth all the way from the seed to the senescence; they are influenced by a plant’s fitness, and they affect its harvest and utilization. Microflora was analyzed by cultivation on specific substrates, total microbial count and five specific microbial groups were observed and quantified. Forage species did not affect plant microflora. The highest risk factor of microbial contamination of feed was proved to be harvest date. Mycotoxin contamination of fresh feed was determined (deoxynivalenol and zearalenone) using ELISA. Zearalenone (ZEA) levels were negatively correlated to fertilization intensity, although these results were not statistically significant. Deoxynivalenol (DON) levels were the lowest in a moderate fertilization regime. Significant differences in mycotoxin content were found among botanical species.

ABSTRACT In this study, we reported the evaluation of ruminal parameters and the rate and degree of degradation of biogenic amines (BA) in vitro, using ruminal fluid from sheep fed two different diets (meadow hay and silage of Medicago sativa L.). Herein, the Charolais sheep breed with a ruminal cannula was used to gather samples. Multiple rumen parameters were evaluated after 10 days of the feeding period. Samples were processed using fermenters inoculated by BA (histamine, tyramine, putrescine, cadaverine, spermine, and spermidine) during 8-h fermentation. Levels of BA were determined using ion-exchange chromatography, and volatile fatty acids (VFA) were determined using gas chromatography. More rapid BA degradation was noticed in hay than in haylage. Overall, VFA levels increased with duration of the fermentation, while pH lowered. The rapid increase was observed after 4 h of fermentation. Ammonia concentration had a decreasing trend in silage and increasing in the hay. Hypotheses on BA degradation were accepted. Important knowledge of the development of ruminal parameters in time was obtained, and based on gathered information, it is possible to say that other parameters and their impacts on BA in rumen need to be examined further.

ABSTRACT: Objectives: This study aimed to detect and characterise methicillin-resistant Staphylococcus aureus (MRSA) from retail meat in the Czech Republic.Methods: Isolates were identified by PCR detection of the S. aureus-specific fragment Sa442 and mecA gene. spa typing, MLST, detection of genes encoding staphylococcal enterotoxins, Panton–Valentine leukocidin (pvl), exfoliative toxins A and B (eta and etb), toxic shock syndrome toxin (tst) and staphylokinase (sak), detection of φSa3 prophage and antimicrobial susceptibility testing were performed.Results: Of 65 raw meat samples examined (poultry, beef, pork and rabbit), 23 (35.4%) were positive for MRSA. Twelve positive samples originated from poultry (12/33; 36.4%), while the remaining eleven came from pork (9/9; 100%) and pork/beef mixed minced meat (2/5; 40.0%). Eight spa types belonging to five different sequence types (STs) were identified. ST398 was the most frequent (28/36; 77.8%), presenting spa types t011, t034, t2576, t4132, t588 and t899. Other livestock-associated MRSA STs (ST9-t899, ST5-t002, ST692-t8646 or the newly described ST4034-t899) were also sporadically identified. In seven isolates (19.4%), one or more staphylococcal enterotoxin genes were detected, with sea, seg and sei prevailing. Three isolates from turkey [ST398-t899 (n = 2) and ST398-t011] harboured the sak gene, and the latter also harboured the sea gene. Seven isolates from poultry harboured the φSa3 prophage and were resistant to tetracycline.Conclusion: Specific kinds of meat appear to be a possible source of MRSA, although the risk to humans is hard to define. Therefore, surveillance of MRSA in meat as well as hygienic practices should be improved.

Sorghum is a forage species important in animal nutrition with an increased potential in the upcoming difficulties in agricultural practices in regard to climate change. Therefore, this paper describes its yield and nutritional parameters depending on growing conditions simulated on two locations (humid and fertile Obora, less fertile and dry Písky), two sowing dates (29. 5. and 25. 6.), and variety (11 varieties). Fresh matter yield was determined by weighing the sample on the field immediately after harvest, Dry matter yield and nutritional content were determined in laboratory by Weende analysis from dried and homogenized samples. Nutritional parameters measured were crude fibre, fat, nitrogen and ash content. Additionally, digestibility of organic as well as dry matter was determined. No significant differences in average fresh matter yield or dry matter yield were found between the two locations and sowing dates in general. There were significant differences among varieties, the highest yields were measured in KWS Zerberus (51.57 ± 3.76 t/ha of fresh matter yield, 15.98 ± 1.34 t/ha of dry matter yield, with higher values observed on Písky). Our data suggest, that sowing date does significantly affect fat content (higher in June sowing). Other nutritional parameters were also higher in sorghum sown in June, with the exception of ash. Nutritional composition of samples did not significantly differ variety to variety, however, there were differences in digestibility of the biomass. Organic matter digestibility was 70.58–85.67%, dry matter digestibility was 73.21–86.70%, with highest digestibility in DMS 45-480. Varieties with the highest importance to farmers in the area are KWS Zerberus, KWS Tarzan and KWS Kallisto (based on dry matter yield), and Triumfo BMR, Sweet Susana and DSM 45-480 (based on digestibility). Sorghum was able to perform well even on dry sandy soil with lower fertilization, which shows the high variability and suitability of sorghum in agricultural practice.

Low plant species richness and abundance, as well as contamination, can lead to a lack of nutrients in the diets of pollinators. This can cause increased oxidative stress, low resistance to disease and reduced detoxification ability. We proposed three forage mixtures with different botanical compositions, all of which should provide continuous forage for a wide range of pollinators. The monitored mixtures were treated by foliar application with selenium and zinc. Both elements should increase the quality of pollen and nectar. The effect of meadow mixtures and microelements on pollinator abundance, richness, and preference was evaluated using data obtained via the line transect method. Furthermore, the amount of selenium and zinc in the flowers was determined. It was found that the microelement treatment did not have any effect on the abundance and richness of the main pollinator taxa, which were affected only by a botanical composition of meadow mixtures. However, a preference for zinc-treated forage was observed in Apis mellifera, and a preference for selenium-treated forage was observed in Bombus spp. These two eusocial taxa appear to respond to an increased amount of microelements in the treated meadow mixtures. On the contrary, Lepidoptera was tied to untreated forage, where they were probably pushed by social bee species due to the competition.

The global food chain may significantly promote the dissemination of bacteria resistant to antibiotics around the world. This study was aimed at determining the prevalence and genetic characteristics of Enterobacteriaceae with mcr-mediated colistin (CT) resistance in retail meat of different origins. Bacteria of the Enterobacteriaceae family carrying the mcr-1 gene were detected in 21% (18/86) of the examined samples, especially in turkey meat and liver originating from EU and non-EU countries (19%) and in rabbit meat imported from China (2%). The examined samples of the meat and liver of chicken and other poultry and of pork and beef were negative for the presence of bacteria carrying the mcr-1 to mcr-5 genes. A huge number of isolates belonging to Escherchia coli (n = 54), Klebsiella pneumoniae (n = 6), and Citrobacter braakii (n = 1) carrying the mcr-1 gene were obtained. Despite the high heterogeneity of the tested isolates, the mcr-1 gene was localized on only three types of plasmids (IncX4, IncHI2, and IncI2). The most frequent type of plasmid was IncX4, which carried the mcr-1 gene in 77% of E. coli and K. pneumoniae isolates from turkey meat and liver samples from the Czechia, Germany, Poland, and Brazil. Our findings indicate highly probable interspecies transfer of IncX4 and IncI2 plasmids within one meat sample. The co-resistance of plasmid-mediated CT resistance encoded by the mcr-1 and ESBL genes was detected in 18% of the isolates. Another noteworthy finding was the fosA3 gene coding for fosfomycin resistance in a multidrug-resistant isolate of E. coli from rabbit meat imported from China. The observed high level of Enterobacteriaceae with plasmids carrying the mcr-1 gene in retail meat reflects the need for Europe-wide monitoring of mcr-mediated CT resistance throughout the whole food chain.

The great plasticity and diversity of the Escherichia coli genome, together with the ubiquitous occurrence, make E. coli a bacterium of world-wide concern. Of particular interest are pathogenic strains and strains harboring antimicrobial resistance genes. Overlapping virulence-associated traits between avian-source E. coli and human extraintestinal pathogenic E. coli (ExPEC) suggest zoonotic potential and safety threat of poultry food products. We analyzed whole-genome sequencing (WGS) data of 46 mcr-1-positive E. coli strains isolated from retail raw meat purchased in the Czech Republic. The investigated strains were characterized by their phylogroup—B1 (43%), A (30%), D (11%), E (7%), F (4%), B2 (2%), C (2%), MLST type, and serotype. A total of 30 multilocus sequence types (STs), of which ST744 was the most common (11%), were identified, with O8 and O89 as the most prevalent serogroups. Using the VirulenceFinder tool, 3 to 26 virulence genes were detected in the examined strains and a total of 7 (15%) strains met the pathogenic criteria for ExPEC. Four strains were defined as UPEC (9%) and 18 (39%) E. coli strains could be classified as APEC. The WGS methods and available on-line tools for their evaluation enable a comprehensive approach to the diagnosis of virulent properties of E. coli strains and represent a suitable and comfortable platform for their detection. Our results show that poultry meat may serve as an important reservoir of strains carrying both virulence and antibiotic resistance genes for animal and human populations.

The microbiological and hygienic quality of cow's, goat's and sheep's milk in the Czech Republic was evaluated. Milk (230 samples) was collected on 41 farms and investigated from May 2012 to October 2014. Milk was analysed for the presence of selected groups and types of bacteria: mesophilic microorganisms (total plate count - TPC), enterococci, Enterobacteriaceae, Escherichia coli, Staphylococcus aureus, Salmonella spp., Campylobacter spp., and Listeria monocytogenes. Besides these indicators and pathogenic agents, somatic cell count was determined as one of the indicators of mammary gland health in cows. TPC ranged between 8.3 × 102 and 1.2 × 109 CFU/ml and somatic cells between 1.6 × 104 and 6.8 × 106 cells/ml. The presence of E. coli was confirmed in 86.3% of samples and the colony counts ranged from 1.0 × 101 to 4.0 × 106 CFU/ml. The presence of verotoxigenic E. coli was confirmed in 3 samples (1.3%) (cow's milk 0%; goat's milk 6.3%; sheep's milk 4.4%). The presence of S. aureus was confirmed in 29.1% of the samples (cow's milk 26.9%; goat's milk 34.4%; sheep's milk 39.1%), but the numbers were very low (< 5.0 × 102 CFU/ml). L. monocytogenes was confirmed in 3 examined samples (1.3%) (cow's milk 0.6%; goat's milk 3.1%; sheep's milk 4.4%). Salmonella spp. and Campylobacter spp. were not detected in any of the samples tested.

This study concentrates on information concerning the microbiological hazards that can be present in raw milk from animal species other than cows. A total of 54 (23 of ovine and 31 of goat) bulk tank milk samples from 10 farms in the Czech Republic were collected in years 2013 - 2014. The sampling was done at regular time intervals during the whole year, with five to eight samples collected from each of the 10 dairy farms involved in the study. All milk samples were collected into sterile sampling bottles and transported in a cooler sampling case to the laboratory for immediate examination. Farms were randomly selected to cover the whole area of the Czech Republic. The prevalence and characteristic of Escherichia coli, Staphylococcus aureus, Salmonella spp., Campylobacter spp. and Listeria monocytogenes was studied. Raw cow's milk can be contaminated by E. coli intramammarily during clinical or subclinical mastitis and either directly through animal feces or indirectly during milk collection through farm employees or the milking equipment. E. coli was detected in 90.3% of the goat milk and 95.7% of the ovine milk samples. The genes encoding Shiga toxins 1 and 2- (stx1, stx2) were not detected and no STEC was identified. The Eae was the detected in 3 (4.6%) isolates. S. aureus was detected in 9 (29.0%) samples of goat milk and 8 (34.8%) samples of ovine milk. A total 12 (57.1%) enterotoxin positive S. aureus were obtained; 6 (28.6%) were positive for the production of sec encoding enterotoxin SEC; in 4 (19.0%) isolates the gene seh was detected; 2 (9.5%) isolates were proven positive for seg (4.8%) and combination seg and sei (4.8%). The presence of MRSA was not detected in the tested samples in our study. L. monocytogenes was detected in 1 (3.2%) samples of goat milk and 1 (4.3%) samples of ovine milk. The serotype (1/2a, 1/2b) was detected in our study. Campylobacter spp. and Salmonella spp. were not isolated from any of the samples. These results form the basis for determining the microbiological quality standards for goat and ovine milk.

There was investigated microbial quality of spices used in production of meat products (black pepper, allspice, coriander, juniper, cumin, cinnamon, badian, mustard, bay leaf, paprika, rosemary, garlic, ginger, thyme, cardamom). The spices were analysed on the presence of total count of mesophilic, thermoresistant and coliforming microorganisms, Staphylococcus aureus, methicilin resistant S. aureus (MRSA), Escherichia coli, Salmonella spp., Bacillus cereus, Bacillus licheniformis and moulds. For the detection of fungal contamination was used agar with glucose, yeast extract and oxytetracyklin and dichloran-glycerol agar. The cultivation was performed at 25 ±1°C for 5 - 7 days. The microscopic method was used for species identification. The aflatoxin presence was confirmed by ELISA test in all of tested spices and was performed in ppb (pars per billion = μg/kg). TCM ranged from 200 to 5600000 cfu/g, TRM from 20 to 90000 cfu/g and coliforming bacteria from 30 to 3200 cfu/g. B. cereus was present in juniper, mustard, bay leaf, thyme and cardamom (32%), while B. licheniformis was confirmed in 58% of cases (allspice, pepper, ground juniper, badian, bay leaf, paprika, garlic, thyme and cardamom). S. aureus was detected in whole coriander, cinnamon, badian and mustard but only in law number (30, 40, 20 and 10 cfu/g respectively). No strains S. aureus was identified as MRSA. The presence of Salmonella spp. and E. coli was not confirmed. The fungal contamination was found in 14 spices and the their count varied from 0 to 1550 cfu/g. There were confirmed the presence of Aspergillus flavus (allspice whole and ground, black pepper whole and ground, whole coriander, ground cumin, ground bay leaf), Aspergillus niger (allspice whole and ground, black pepper ground, ground juniper, cumin ground, bay leaf ground, ground rosemary, ground thyme), Penicillium glaucum (allspice whole and ground, whole juniper, whole cinnamon), Penicillium claviforme (whole black pepper, whole coriander, cardamom ground), Alternaria alternata (cumin ground, rosemary ground, thyme ground), Mucor (whole and ground coriander and thyme) and Phoma (ground cumin). The aflatoxin presence was confirmed in 11 of samples (57.9%) and the value ranged from 0 to 4 ppb (ground allspice, whole and ground pepper, whole juniper, cumin, cinnamon, badian, bay leaf, paprika, rosemary, thyme).

A meat paste production line and its microbial parameters have been evaluated in single Czech company. The raw meat paste samples before heat treatment were tested positively for the presence of three staphylococci species: Staphylococcus aureus, Staphylococcus haemolyticus and Staphylococcus epidermidis. Subsequent microbial analysis of meat paste components and ingredients (fresh meat, water, spices, equipment) identified only the spices used as positive for S. aureus (coriander, cinnamon, badian, mustard – (10 - 40 cfu/g)) and S. haemolyticus strains (juniper, ginger). The collection of sixteen collected strains (S. aureus (n = 4), S. haemolyticus (n = 4), S. epidermidis (n = 8)) has been typed with the rep-PCR method utilising (GTG)5 primer. Analysis of the fingerprints using the unweighted pair-group method using arithmetic averages (UPGMA) clustering method revealed presence of eleven strain clusters with similarity lower than 90%: two fingerprint clusters of S. aureus, three individual clusters characteristic for S. haemolyticus and six different S. epidermidis specific clusters. The S. aureus strains from different types of spice were identical, resp. very similar. Molecular tracking composed from the rep-PCR analysis of acquired isolates and comparison among all collected fingerprints confirmed the spices to be the source of both S. aureus and S. haemolyticus strains identified in raw meat paste. The additional rep-PCR analysis of the S. epidermidis collection confirmed usability and performance of this method. The antibiotic susceptibility to fourteen individual antibiotics has been examined among the collected staphylococci strains. The predominant erythromycin resistance (68.8%) was followed with the resistance to amoxicillin/clavulanic acid (56.2%). Other resistances observed were less frequent (clindamycin – 12.5%, oxacillin – 6.3%, tetracycline – 6.3%, sulphamethoxazole-trimethoprim – 6.3%, chloramphenicol – 6.3%, novobiocin – 6.3%). As shown by our experimental results, rep-PCR with the (GTG)5 primer is an applicable tool for typing of bacterial strains and may be used for identifying the source of contamination.