The aim of the study was the assessment of microbiological quality of compound feed used in Poland in 2007-2010. The examinations were done at all veterinary diagnostic laboratories operating in the frame of official laboratory system. The occurrence of Salmonella sp. and counts of Enterobacteriaceae family, mesophilic aerobic bacteria, total microorganisms, and fungi were assessed. Assays were done following Polish, European, and international standards. Percentage of contamination of compound feed for poultry, pigs, and cattle by Salmonella sp. ranged from 0% to 3.5%. The highest contamination level by Enterobacteriaceae bacteria were detected in wet petfood. No more than 106 cfu/g of aerobic bacteria and no more than 105 cfu/g of fungi were detected in the feed. The results of the study revealed that the microbiological quality of compound feed used in Poland in 2007-2010 was better than the quality of the feed used in 2003-2006.

The aim of this study was the evaluation of the insect processed animal protein (IPAP) contamination level by Clostridium spp. Particularly, we screened for the occurrence of pathogenic species of Clostridia. The samples of IPAP were derived from yellow mealworm (Tenebrio molitor) and black soldier fly (Hermetia illucens) available in the Polish market. The IPAPs were added to experimental feeds for poultry. The differences between the contamination levels of the control (without the addition of IPAP) and experimental (with the addition of IPAP) groups were monitored. The samples were also examined by culture and PCR-based methods to detect 16S rDNA and genes determining botulinum toxin (BoNT) production. Statistical significance was noticed among the feed with the IPAP addition, as well as an increase of contamination by Clostridium spp. In one sample of IPAP, the occurrence of ntnh and bont/D genes determining the production of BoNT/D was noticed. However, a positive result was noticed only at the step of the liquid culture; the Clostridium botulinum type D strain was not isolated. Phenotypically, and according to the 16S rDNA analysis, genetically similar strains to C. botulinum species were isolated. Considering the microbiological safety of IPAP and expanding possibility of its use in livestock animal feed, it seems to be reasonable to provide complex risk assessment on the potential transfer of Clostridia into feed compounds, to assure the safety and sustainable development of insect PAP industry.

The aim of this study was to assess occurrence of Clostridium botulinum and Clostridium perfringens in honey samples from Kazakhstan. Analyses were carried out using a set of PCR methods for identification of anaerobic bacteria, and detection of toxin genes of C. botulinum and C. perfringens. Among 197 samples, C. botulinum was noticed in only one (0.5%). The isolated strain of this pathogen showed the presence of the bont/A and ntnh genes. C. perfringens strains were isolated from 18 (9%) samples, and mPCR (multiplex PCR) analysis led to them all being classified as toxin type A with the ability to produce α toxin. Sequence analysis of 16S rDNA genes showed occurrence in 4 samples of other anaerobes related to C. botulinum, which were C. sporogenes and C. beijerinckii strains. C. botulinum prevalence in honey samples from Kazakhstan in comparison to the prevalence in samples collected from the other regions seems to be less. The highest prevalence of Clostridium sp. was noticed in the East Kazakhstan province. Our study is the first survey on BoNT-producing clostridia and C. perfringens prevalence in Kazakh honey.

The paper presents the first results of a study on the contamination of honey produced in the Republic of Kazakhstan with C. botulinum spores known to pose a potential infection threat to infants. During microbiological analysis, culturing methods with TPGY, Willis-Hobbs agar, FAA agar connected with PCR, sequencing, and a mouse bioassay were used. The C. botulinum contamination rate of honey was relatively low as determined, at 0.91%. Nonetheless, the potential danger of the bacteria to childrens' health should not be neglected.

The aim of this study was to perform an in-house validation of multiplex PCR method for C. botulinum detection in food and feed samples. The study was carried out on food and feed matrixes artificially contaminated by spores of C. botulinum reference strains. The following characteristic parameters for qualitative detection were estimated: limit of detection expressed as LOD 50 according to the Spearman-Kärber formula, specificity, sensitivity, and accuracy according to the PN-EN ISO 16140:2004. The validated method showed high specificity. Specific PCR products were revealed only for DNA obtained from samples contaminated with C. botulinum spores. PCR inhibition was observed, especially during examination of contaminated feed. The calculated LOD 50 for feed was nearly 10 times higher than for food. The implemented method enables to obtain test results during 3 d without timeconsuming process of isolation and proving the ability of strains to produce botulinum toxins.

The aim of this study was to assess the possibility of genetically modified DNA transfer from feed containing RR soybean or/and MON810 maize to animal tissues, gut bacterial flora, food of animal origin, and the fate of GM DNA in the animal digestive tract. The experiment was carried out on broilers, laying hens, pigs and calves. All animals were divided into four groups: I -control group (non-modified feed), II -GM soybean group (non-modified maize, RR soybean), III -GM maize group (MON810 maize, non-modified soybean), and IV -GM maize and soybean group (MON810 maize, RR soybean). Samples of blood, organs, tissues, digesta from the gastrointestinal tract, and eggs were analysed for the presence of plant species specific genes, and transgenic sequences of CaMV 35S promoter and NOS terminator. PCR amplifications of these GM sequences were conducted to investigate the GM DNA transfer from feed to animal tissues and bacterial gut flora. In none of the analysed samples of blood, organs, tissues, eggs, excreta and bacterial DNA were plant reference genes or GM DNA found. A GM crop diet did not affect bacterial gut flora as regards diversity of bacteria species, quantity of particular bacteria species in the animal gut, or incorporation of transgenic DNA to the bacteria genome. It can be concluded that MON810 maize and RR soybean used for animal feeding are substantially equivalent to their conventional counterparts. Genetically modified DNA from MON810 maize and RR soybean is digested in the same way as plant DNA, with no probability of its transfer to animal tissues or gut bacterial flora.

IntroductionSilage 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 MethodsAnalyses 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.ResultsThe 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.ConclusionsDeterioration 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.

IntroductionThe aim of this study was examination of honey samples collected from apiaries situated in all Polish provinces for occurrence of Clostridium spp., especially C. perfringens.Material and MethodsThe study was carried out on 240 honey samples (15 samples/province). Estimation of Clostridium titre, its cultures and C. perfringens isolate characterisation were performed according to the standard PN-R-64791:1994. A multiplex PCR method for detection of genes coding cpa (α toxin), cpb (β), cpb2 (β2), etx (ε), iap (ι), and cpe (enterotoxin) toxins was used.ResultsClostridium spp. was noticed in 56% (136/240) of samples, and its titres ranged between 0.1 g and 0.001 g. Clostridium perfringens occurrence was evidenced in 27.5% (66/240) of samples. All isolates were classified to toxinotype A.ConclusionsEvidence of a high number of positive samples with occurrence of Clostridium spp. indicates a potential risk to consumers’ health. The infective number of Clostridium spp. is unknown; however, the obtained results have shown that a risk assessment on the entire honey harvesting process should be made in order to ensure microbiological safety. Moreover, a detailed study should be undertaken on the antibiotic resistance of C. perfringens isolates from honey samples.