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.

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.

The diversity of BoNT-producing Clostridia is still a worrying problem for specialists who explore the evolutionary and taxonomic diversity of C. botulinum. It is also a problem for epidemiologists and laboratory staff conducting investigations into foodborne botulism in humans and animals, because their genetic and phenotypic heterogeneity cause complications in choosing the proper analytical tools and in reliably interpreting results. Botulinum neurotoxins (BoNTs) are produced by several bacterial groups that meet all the criteria of distinct species. Despite this, the historical designation of C. botulinum as the one species that produces botulinum toxins is still exploited. New genetic tools such as whole-genome sequencing (WGS) indicate horizontal gene transfer and the occurrence of botulinum gene clusters that are not limited only to Clostridium spp., but also to Gram-negative aerobic species. The literature data regarding the mentioned heterogeneity of BoNT-producing Clostridia indicate the requirement to reclassify C. botulinum species and other microorganisms able to produce BoNTs or possessing botulinum-like gene clusters. The aim of this study was to present the problem of the diversity of BoNT-producing Clostridia over time and new trends toward obtaining a reliable classification of these microorganisms, based on a complex review of the literature.

The following should be added to the Acknowledgments section of this paper [...]