The Identification and Quantification of 21 Antibacterial Substances by LC-MS/MS in Natural and Organic Liquid Fertilizer Samples.

Antibiotics in animal production are widely used around the world for therapeutic and preventive purposes, and in some countries, they still serve as antibiotic growth stimulants. Regardless of the purpose of using antibiotics in livestock, they may be present in animal tissues and organs as well as in body fluids and excretions (feces and urine). Farm animal excrement in unprocessed form (natural fertilizers) or processed form (organic fertilizers) is applied to agricultural fields because it improves soil fertility. Antibiotics present in fertilizers may therefore contaminate the soil, surface, groundwater, and plants, which may pose a threat to the environment, animals, and humans. Therefore, it is important to develop analytical methods that will allow for the control of the presence of antibacterial substances in natural and organic fertilizers. Therefore, in this study, an LC-MS/MS method was developed and validated for the determination of 21 antibacterial substances in natural and organic liquid fertilizers. The developed method was used to analyze 62 samples of natural and organic liquid fertilizers, showing that over 24% of the tested samples were contaminated with antibiotics, mainly from the group of tetracyclines and fluoroquinolones. Studies of post-fermentation sludge from biogas plants have shown that the processes of anaerobic methane fermentation, pH, and temperature changes taking place in bioreactors do not lead to the complete degradation of antibiotics present in the material used for biogas production. For this reason, monitoring studies of natural and organic fertilizers should be undertaken to limit the introduction of antibiotics into the natural environment.

Simultaneous determination of sulfonamides, trimethoprim, amoxicillin and tylosin in medicated feed by high performance liquid chromatography with mass spectrometry.

Introduction: The article presents a rapid and simple analytical procedure for determination of four sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine and sulfamethoxazole), trimethoprim, tylosin and amoxicillin in animal medicated feed. Material and Methods: Eighteen medicated feed samples were analysed for active substances. The analytical protocol used a mixture of acetonitrile and 0.05 M phosphoric buffer, pH 4.5 for the extraction of seven antibacterial substances. After extraction, the samples were diluted in Milli-Q water and analysed by liquid chromatography with mass spectrometry. The developed procedure was subjected to validation in terms of linearity, selectivity, limits of quantification and determination, repeatability, reproducibility and uncertainty. Results: The validation of the method was carried out in accordance with the criteria set out in Commission Implementing Regulation (EU) 2021/808 and ICH guidelines. This method provided average recoveries of 90.8 to 104.5% with coefficients of variation for repeatability and reproducibility in the ranges of 3.2-6.9% and 5.2-8.3%, respectively for all analysed antibacterial substances. The limit of detection and limit of quantification for all seven analytes ranged from 5.4 mg/kg to 48.3 mg/kg and from 10.4 mg/kg to 119.3 mg/kg, respectively. The uncertainty of the method depending on the compound varied from 14.0% to 24.0%. The validated method was successfully applied to the 18 medicated feeds. Conclusion: The developed method can be successfully used to routinely control the content and homogeneity of seven antibacterial substances in medicated feed.

Development, in-house validation and application of a method using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) for the quantification of 12 ergot alkaloids in compound feeds.

Introduction: Ergot alkaloids (EAs) are toxic substances naturally produced by Claviceps fungi. These fungi infest a wide range of cereals and grasses. When domestic animals are exposed to EAs through contaminated feeds, it is detrimental to them and leads to significant economic losses. For that reason, it is important to monitor feed for the presence of EAs, especially with methods enabling their determination in processed materials. Material and Methods: Ergot alkaloids were extracted with acetonitrile, and dispersive solid phase extraction (d-SPE) was used for clean-up of the extracts. After evaporation, the extracts were reconstituted in ammonium carbonate and acetonitrile and subjected to instrumental analysis using high-performance liquid chromatography with fluorescence detection. The developed method was validated in terms of linearity, selectivity, repeatability, reproducibility, robustness, matrix effect, limits of quantification and detection and uncertainty. The EA content of 40 compound feeds was determined. Results: All the assessed validation parameters fulfilled the requirements of Regulation (EU) 2021/808. At least one of the monitored alkaloids was determined in 40% of the samples. The EAs with the highest incidence rate were ergocryptine, ergometrinine and ergocornine. The total concentrations of EAs ranged from under the limit of quantification to 62.3 µg kg-1. Conclusion: The results demonstrated that the developed method was suitable for simultaneously determining twelve EAs in compound feed and could be used for routine analysis.

Clostridium perfringens-Opportunistic Foodborne Pathogen, Its Diversity and Epidemiological Significance.

The C. perfringens species is associated with various environments, such as soils, sewage, and food. However, it is also a component of the gastrointestinal (GI) microflora (i.e., microbiota) of sick and healthy humans and animals. C. perfringens is linked with different systemic and enteric diseases in livestock and humans, such as gas gangrene, food poisoning, non-foodborne diarrhoea, and enterocolitis. The strains of this opportunistic pathogen are known to secrete over 20 identified toxins that are considered its principal virulence factors. C. perfringens belongs to the anaerobic bacteria community but can also survive in the presence of oxygen. The short time between generations, the multi-production capability of toxins and heat-resistant spores, the location of many virulence genes on mobile genetic elements, and the inhabitance of this opportunistic pathogen in different ecological niches make C. perfringens a very important microorganism for public health protection. The epidemiological evidence for the association of these strains with C. perfringens-meditated food poisoning and some cases of non-foodborne diseases is very clear and well-documented. However, the genetic diversity and physiology of C. perfringens should still be studied in order to confirm the importance of suspected novel virulence traits. A very significant problem is the growing antibiotic resistance of C. perfringens strains. The aim of this review is to show the current basic information about the toxins, epidemiology, and genetic and molecular diversity of this opportunistic pathogen.

Insect Meals and Insect Antimicrobial Peptides as an Alternative for Antibiotics and Growth Promoters in Livestock Production.

The extensive use of antibiotics in animal production has led to the development of antibiotic-resistant microorganisms and the search for alternative antimicrobial agents in animal production. One such compound may be antimicrobial peptides (AMPs), which are characterized by, among others, a wide range of biocidal activity. According to scientific data, insects produce the largest number of antimicrobial peptides, and the changing EU legislation has allowed processed animal protein derived from insects to be used in feed for farm animals, which, in addition to a protein supplement, may prove to be an alternative to antibiotics and antibiotic growth promoters due to their documented beneficial impact on livestock health. In animals that were fed feeds with the addition of insect meals, changes in their intestinal microbiota, strengthened immunity, and increased antibacterial activity were confirmed to be positive effects obtained thanks to the insect diet. This paper reviews the literature on sources of antibacterial peptides and the mechanism of action of these compounds, with particular emphasis on insect antibacterial peptides and their potential impact on animal health, and legal regulations related to the use of insect meals in animal nutrition.

Prevalence of veterinary antibiotics in natural and organic fertilizers from animal food production and assessment of their potential ecological risk.

BACKGROUND: Veterinary antibiotics are emerging contaminants and enter into soil principally by agricultural application of organic fertilizers. This article presents the results of the research obtained for the analyzed 70 samples of fertilizers (pig and poultry manure and slurry and digestate) for various classes of antibiotics. RESULTS: Doxycycline, oxytetracycline, tetracycline, lincomycin, tiamulin and enrofloxacin were found in tested samples. Doxycycline was found as a dominant compound, and its highest concentration was 175 mg/kg in pig manure. This investigation indicated that fertilization with manure, especially animal feces, might be the primary source of antibiotics. Additionally, a risk assessment based on a risk quotient was carried out, which showed that the determined concentrations of antibiotics in fertilizers may pose a threat to soil microorganisms. CONCLUSIONS: Results suggested that the ecological risk effects of antibiotic contamination on soil bases and their potential adverse risk on human health needs special attention. © 2023 Society of Chemical Industry.

Laboratory Experience with the Microscopic Method for the Detection of Insects in Poultry Feeds.

Introduction: The use of insects and their processed animal proteins (PAPs) for animal nutrition creates the need for research into methods useful for routine surveillance for their presence. The aim of this study was to evaluate a modified microscopic method for the detection of particles of insects in poultry feed. Material and Methods: A total of 90 samples including PAP of insects (Hermetia illucens and Tenebrio molitor), poultry feeds produced with different levels (0-27%) of insect PAP content, and other poultry feeds spiked with insect PAP at 1% were investigated using a modified microscopic method with a double sedimentation protocol. Results: Characteristic features of insects including cuticulae, muscles, bristles and tracheoles were determined in the microscopic images obtained. In all spiked samples, characteristic fragments of insects were detected. The fragments of muscle and tracheoles only indicated the presence of material from members of the insect class but could not facilitate identification of organisms to species level. Conclusion: The results obtained with this double sedimentation protocol for the isolation of insect PAP from feed for poultry have shown that the method can be used in routine analysis.

Development and application of criteria for classification of hydrolysed proteins in the framework of feed safety.

In the view of a circular economy, there is an increasing need for (re-)using animal by-products that have a wide range of applications and sufficient safety. Hydrolysates of animal proteins (HPs) are frequently used as feed ingredients. Nevertheless, clear criteria for legal use and methods for monitoring feed applications are not available. Here, a range of methods have been used and evaluated for characterizing a set of 26 samples of hydrolysed proteins, 'hydrolysed' feather meals and processed animal proteins (PAPs), with verification based on an additional set of eight samples. Methods included determination of ash content, sediment (mineral fraction) content, protein content, species identity, solubility, protein solubility, size exclusion chromatography and polyacrylamide gel electrophoresis (SDS-PAGE). After a comparison of results obtained with water and SDS, water was chosen as the solvent for environmental and occupational reasons. Typical HP samples have a protein content higher than 60%, a solubility exceeding 50% and a virtual absence of a mineral fraction. The first discrimination between HPs and PAPs could be based on the absence or presence, respectively, of a mineral fraction. An approach for HP characterization is designed using a Hydrolysation Index (HI) based on the fraction of peptides smaller than 10 kDa, the solubility of the sample and the fraction of soluble proteins. A simplified version (HIs), exclusively based on the fraction of peptides smaller than 10 kDa and the solubility of the sample, shows a trend among the samples highly comparable to HI. Values for HI and HIs exceeding 60% would characterise HPs. Feather meals, which are heat treated instead of treatment by a chemical process of hydrolysation, range among the PAPs and should not be indicated as "hydrolysed." The HIs can be used as an easy parameter for classifying HPs and for legal enforcement.

Simultaneous Determination of Pyrrolizidine and Tropane Alkaloids in Honey by Liquid Chromatography-mass Spectrometry.

Introduction: Pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) are natural contaminants of honey and respectively hepatoxic and neurotoxic compounds. Because honey is a popular constituent of the human diet, it is relevant to warrant the safety of the product. For that reason, a method for simultaneous determination of PAs and TAs in honey based on liquid chromatography- mass spectrometry was developed. Material and Methods: The analytical protocol used sulphuric acid extraction and solid-phase extraction purification. The developed procedure was subjected to validation in terms of linearity, selectivity, repeatability, reproducibility, limits of quantification and determination, matrix effect and uncertainty. A total of 29 honey samples were analysed for the determination of PAs and TAs. Results: All the evaluated validation parameters fulfilled the requirements of European Commission Decision 2002/657/EC. At least one of the monitored alkaloids was determined in 52% of the samples. Among the most abundant alkaloids were echimidine, intermedine and lycopsamine. The total PA concentrations ranged from 2.2 to 147.0 µg kg-1. Contrastingly, none of the monitored TAs was detected in the analysed samples. An assessment of the dietary exposure to PAs from the consumption of the contaminated honeys showed that three of them would pose a risk to consumers, especially if they were children. Conclusion: A sensitive method suitable for simultaneous determination of PAs and TAs in honey was developed and validated. The analysis of 29 honey samples for PAs and TAs revealed that honey destined for retail could pose a risk to consumers.

Animal Botulism in Poland - Laboratory and Epidemiological Investigations.

Introduction: The aim of the study was to present cases of botulism in animals found in Poland in 2019-2021. The analytical laboratory diagnosis and difficulties that occurred in the interpretation of the results are described. Material and Methods: From 2019 to 2021, samples of serum, intestinal content, liver, spleen, kidney, faeces, wet feed, dry feed, ensilage, water and mixed samples of internal organs associated with 10 suspected animal botulism cases were sent to the National Veterinary Research Institute. Samples were analysed using a mouse bioassay and culture methods in combination with ntnh and bont gene detection. Results: Among the ten putative botulism cases, only four (40%) were confirmed in the laboratory on the basis of the detection of botulinum toxin (BoNT) or the ntnh or bont genes. The remaining six (60%) were determined as probable despite observable characteristic clinical signs. Conclusion: The diagnosis of botulism in animals is a very difficult task, made so by the heterogeneity of Clostridium botulinum strains and possible loss of toxinogenicity during laboratory processing or the potential degradation of toxins. Laboratory diagnosis is a complex and problematic process which should utilise different prescribed methods for specific types of sample.

Probiotic Potential of Clostridium spp.-Advantages and Doubts.

Clostridium spp. is a large genus of obligate anaerobes and is an extremely heterogeneous group of bacteria that can be classified into 19 clusters. Genetic analyses based on the next-generation sequencing of 16S rRNA genes and metagenome analyses conducted on human feces, mucosal biopsies, and luminal content have shown that the three main groups of strict extremophile anaerobes present in the intestines are Clostridium cluster IV (also known as the Clostridium leptum group), Clostridium cluster XIVa (also known as the Clostridium coccoides group) and Bacteroides. In addition to the mentioned clusters, some C. butyricum strains are also considered beneficial for human health. Moreover, this bacterium has been widely used as a probiotic in Asia (particularly in Japan, Korea, and China). The mentioned commensal Clostridia are involved in the regulation and maintenance of all intestinal functions. In the literature, the development processes of new therapies are described based on commensal Clostridia activity. In addition, some Clostridia are associated with pathogenic processes. Some C. butyricum strains detected in stool samples are involved in botulism cases and have also been implicated in severe diseases such as infant botulism and necrotizing enterocolitis in preterm neonates. The aim of this study is to review reports on the possibility of using Clostridium strains as probiotics, consider their positive impact on human health, and identify the risks associated with the expression of their pathogenic properties.

Enterococci-Involvement in Pathogenesis and Therapeutic Potential in Cancer Treatment: A Mini-Review.

Enterococcus spp. are Gram-positive, heterogeneous lactic acid bacteria inhabiting various environments. Several species of Enterococci are considered to be able to stimulate the immune system and play an important role in intestinal homeostasis. Some Enterococci can be used as probiotics. Some strains of E. faecium are components of pharmaceutical products used to treat diarrhea, antibiotic-associated diarrhea, or irritable bowel syndrome (IBS). However, it has been proved that they are responsible for food contamination, and are sometimes undesirable from the point of view of food technology. Additionally, the virulence and multi-drug resistance of Enterococci potentially pose a risk of an epidemic, especially in hospital environments. Moreover, there are indications of their negative role in colon tumorigenesis; however, some nterococci are proved to support immunotherapy in cancer treatment. In general, it can be concluded that this group of microorganisms, despite its nature, has properties that can be used to support cancer treatment-both aggressive chemotherapy and cutting-edge therapy targeting immune checkpoints (IC).

HPLC-FLD-Based Method for the Detection of Sulfonamides in Organic Fertilizers Collected from Poland.

Antibacterial substances such as sulfonamides are widely used in veterinary medicine to treat many bacterial diseases. After their administration to animals, up to 90% of the initial dose of the antibiotic is excreted in the feces and/or urine, which can be applied to farmland as natural or organic fertilizers. In this work, an analytical method was developed with the use of HPLC-FLD for the detection and quantification of five sulfonamides (sulfaguanidine, sulfadiazine, sulfamerazine, sulamethazine and sulfamethoxazol) in poultry and pig feces, slurry and digestates. The method was validated according to EU requirements (Commission Decision 2002/657/EC and VICH GL49). Linearity, decision limit, detection capability, detection and quantification limits, recovery, precision, and selectivity were determined, and adequate results were obtained. Using the HPLC-FLD method for all analyzed matrices, recoveries were satisfactory (77.00-121.16%), with repeatability and reproducibility in the range of 4.36-17.34% to 7.94-18.55%, respectively. Decision limit (CCα) and detection capability (CCß) were 33.87-67.63 and 53.36-92.00 µg/kg, respectively, and limit of detection (LOD) and limit of quantification (LOQ) were 13.53-23.30 and 26.02-40.38 µg/kg, respectively, depending on the analyte. The forty-four samples of natural and organic fertilizers were analyzed, and four samples showed sulfamethoxazole in the amount from range 158 to 11,070 µg/kg. The application of antibiotics including sulfonamides for farming animals is widespread and may lead to the development of antibiotic resistance and other environmental effects.

Scopolamine and atropine in feeds - determination with liquid chromatography mass spectrometry.

Tropane alkaloids (TAs) are naturally occurring plant toxins. Due to the fact that TA-producing plants can enter the food chain, they pose a risk for animals and human health. Therefore, sensitive analytical methods need to be developed to provide an adequate safety of feed and food. The presented method is based on liquid chromatography-mass spectrometry detection and enables the determination of scopolamine and atropine in compound feeds at a low level of contamination. Limits of quantification for scopolamine and atropine were 0.92 and 0.93 µg kg-1, respectively. Scopolamine-D3 and atropine-D3 were used for quantification. The method was successfully validated and applied to the analysis of 42 feed samples. Among investigated feeds, 67% contained at least one of the monitored alkaloids. Soybean meals were the feed materials contaminated most often, also with the highest determined concentrations of TAs, which reached 147.9 µg kg-1.

Botulinum toxin in cancer therapy-current perspectives and limitations.

Different serotypes of botulinum toxins (BoNTs) act upon different types of SNARE proteins. This property is used in aesthetic medicine to treat certain eye disorders such as crossed eyes (strabismus) and uncontrolled blinking (blepharospasm), to treat muscle spasms or movement disorders, and, for the two last decades, more and more often, to provide support in cancer therapy, especially so as to obtain analgesic effects upon spastic conditions. The limited literature data also suggests that the addition of BoNTs to the culture of cancer cell lines reduces cell growth, and mitotic activity, and promotes their apoptosis. BoNTs have several advantages that can be emphasized: BoNTs act on both perfusion and oxygenation; moreover, BoNTs are considered to be safe and free of systemic side effects upon administration. Recently, advances in molecular biology techniques have allowed a wide variety of novel BoNT constructs with alternative functions. These constructs could be assessed as potential new classes of anti-cancer drugs. This creates new potential perspectives in the wider use of non-toxic modified BoNT constructs in cancer therapy. In the light of the mentioned premises and existing literature reports, the aim of this review is to summarize current data and reports considering BoNT use in cancer therapy. KEY POINTS : •Botulinum toxin (BoNTs) may be useful in cancer treatment. •Botulinum toxin can serve as an analgesic after cancer radiotherapy. •Botulinum toxin has the ability to inhibit tumor growth and promote apoptosis of neoplastic cells.

Potential Risk of Botulinum Neurotoxin -producing Clostridia Occurrence in Canned Fish.

Introduction: Heat treatment is indispensable in fish canning to provide an acceptable shelf life. Its optimisation reduces the risk of the presence of Clostridium botulinum spores, which could potentially cause botulism cases. This study evaluated canned fish samples for botulism neurotoxin (BoNT)-producing clostridia contamination and can bulging through microbiological contaminant growth. A new analytical approach was developed for detection of such clostridia and phenotypically similar species. Material and Methods: A total of 70 canned fish samples suspected of exhibiting bulging features were analysed. Culture methods were used to detect clostridia. The isolates obtained were evaluated on the basis of the exhibited phenotypic characteristics. Also, PCRs were used for the detection of genes determining BoNT production (non-toxic non-haemagglutinin (ntnh) genes) and the amplification of conservative 16S rDNA genes, which were Sanger sequenced. The obtained sequences were analysed using the Basic Local Alignment Search Tool. Results: Clostridium genus species were isolated from 17 (24%) bulging and organoleptically changed samples. No ntnh genes were present in these isolates; however, sequencing confirmed the presence of C. sporogenes, a species with close affinity to C. botulinum. Conclusion: To eliminate the threat of foodborne botulism, laboratory diagnostic techniques must detect species of the Clostridium genus and elucidate their ability to produce BoNTs. Although Clostridium botulinum is the most common cause of botulism, the possibility may not be ignored that non-pathogenic Clostridium species may acquire botulinum toxigenicity. The similarity between the isolated strains of C. sporogenes and C. botulinum should be incorporated in the optimisation of heat treatment to guarantee a sterilised, microbiologically safe product.

Application of Micellar Mobile Phase for Quantification of Sulfonamides in Medicated Feeds by HPLC-DAD.

Rapid chromatographic procedure for quantification of five sulfonamides in medicated feeds are proposed. Satisfactory separation of sulfonamides from medicated feeds was achieved using a Zorbax Eclipse XDB C18 column (4.6 × 150 mm, 5 µm particle size) with a micellar mobile phase consisting of 0.05 M sodium dodecyl sulphate, 0.02 M phosphate buffer, and 6% propan-2-ol (pH 3). UV quantitation was set at 260 nm. The proposed procedure allows the determination of sulfaguanidine, sulfadiazine, sulfamerazine, sulfamethazine, and sulfamethoxazole in medicated feeds for pigs and poultry. Application of the proposed method to the analysis of five pharmaceuticals gave recoveries between 72.7% to 94.7% and coefficients of variations for repeatability and reproducibility between 2.9% to 9.8% respectively, in the range of 200 to 2000 mg/kg sulfonamides in feeds. Limit of detection and limit of quantification were 32.7-56.3 and 54.8-98.4 mg/kg, respectively, depending on the analyte. The proposed procedure for the quantification of sulfonamides is simple, rapid, sensitive, free from interferences and suitable for the routine control of feeds. In the world literature, we did not find the described method of quantitative determination of sulfonamides in medicated feeds with the use of micellar liquid chromatography.

Prevalence of Genetically Modified Soybean in Animal Feedingstuffs in Poland.

INTRODUCTION: Globally, genetically modified (GM) crops were grown on 191.7 million hectares in 2018, which were mostly sown with soybean, maize, cotton, oilseed rape, and rice. The most popular traits introduced through genetic modification include herbicide and pest insect resistance. The aim of this study was to identify and quantify genetically modified soybean used in animal feed in Poland. MATERIAL AND METHODS: This research was based on the real-time PCR technique. All methods for GM soybean events were adopted from the EURL GMFF database of methods and previously verified to meet the minimum criteria of acceptance. Over 15 years of research, 665 samples were examined in total. RESULTS: The most common GM soybean event was MON40-3-2, tested for from the beginning of the investigation. Next, in decreasing order of frequency, were MON89788, MON87701, and A2704-12. In the majority of samples (606; 91%) GM soybeans were identified at a content level above the 0.9% GM content threshold for mandatory labelling. Only 59 soybean samples (9%) were identified as GM negative. GM negative results were mainly identified during the analyses in the last three years of the study, from 2017 to 2019. CONCLUSION: Our data clearly indicate that the majority of soybean used in Poland for animal feeding was genetically modified.

Analytical capabilities of micellar liquid chromatography and application to residue and contaminant analysis: A review.

Green chemistry is the use of chemistry to reduce or eliminate the use of generation of feedstocks, products, by-products, solvents, reagents, etc. that are hazardous to human health or the environment. One of the branches of green chemistry is micellar liquid chromatography. Micellar liquid chromatography is a reversed-phase liquid chromatographic mode with mobile phases containing a surfactant above its critical micellar concentration. The applications of micellar liquid chromatography for the determination of numerous compounds in pharmaceutical formulation, biological samples, food, environmental samples, and feeds have been growing rapidly. Micellar liquid chromatography technique has several advantages over other chromatographic techniques. Its main advantage is the small amount of organic modifiers used such as acetonitrile and methanol and the safety and recyclability of the mobile phase. In our work, we discuss the development of "green chemistry" and present what micellar liquid chromatography is. This article presents application methods with the use of micellar liquid chromatography for analysis on antibacterial substances, melamine, biogenic amines, plant protection products, flavonoids, as well as peptides in biological matrices such as milk, eggs, tissues, honey, and feed.

Potential Determinants of Clostridium Spp. Occurrence in Polish Silage.

INTRODUCTION: Silage quality deteriorates with Clostridium spp. contamination, and if consumed, such silage jeopardises herd health and productivity. Minimising its occurrence reduces economic and animal welfare risks. The study investigated the influence of environmental and technological determinants on the Clostridium genus' occurrence in silage. MATERIAL AND METHODS: Analyses were conducted on 305 silage samples directly collected from farms located in all Polish provinces. Cultures and isolates were evaluated phenotypically and examined for occurrence of Clostridium spp., particularly C. perfringens and C. botulinum using PCR techniques. The results were statistically analysed using the ᵡ2 test for continuous and Student's t-test for non-continuous values. RESULTS: The most influential effect on Clostridium spp. occurrence is exerted by factors potentially associated with primary production, like the type of fertilisation and the contamination level of the ensiled feed material. Clostridium spp. was detected in 232 (76%) samples, and C. perfringens strains, predominantly toxinotype A, in 79 (26%). C. botulinum occurrence was not detected. CONCLUSIONS: Deterioration of silage by clostridia could be prevented by a properly conducted ensiling process with the addition of starter cultures, but the presence of spores mainly depends on primary production and the extent of contamination of the feed material.