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.

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.

As the test material mink feed with natural microflora was used. The analyses were conducted using Wrzosek and TPGY broth media, and Willis-Hobbs and Zeissler differential agar media. Wrzosek, Willis-Hobbs, and Zeissler media are described in Polish Standards approved by the National Standards Body in Poland and routinely used in detection of anaerobic bacteria in Poland. Detection and identification of C. botulinum was performed with a previously validated real-time PCR method based on ntnh gene detection, which is common in all C. botulinum toxotypes. The use of Wrzosek broth and Zeissler agar in routine analyses for detection and identification of C. botulinum was ineffective and limited. The obtained results showed the highest culturing process effectiveness in TPGY broth with 72 h incubation at 30°C and isolation on Willis-Hobbs agar. The real-time PCR method based on ntnh gene detection used in this study could be utilised as a supplementary tool to the mouse lethality assay.

Clostridium perfringens is one of the most widespread anaerobic spore forming bacteria found in the environment. The toxotype A of the species inhabits the gastrointestinal tract of birds and mammals exhibiting pathogenic properties in the immunocompromised host. The virulence determinants of C. perfringens are toxins and extracellular enzymes which cause gas gangrene, enteritis necroticans, food poisoning, and non-food borne gastrointestinal infections in humans. The young animals suffer from enterotoxaemia, dysentery and necrotic enteritis due to the anaerobic spore forming bacilli. This article reviews the epidemiological significance of C. perfringens and its disease diagnostics, taking into account all known to date virulence determinants of the microorganism.