SUMMARY: The quantification of messenger RNA expression levels by real‐time reverse‐transcription polymerase chain reaction requires the availability of reference genes that are stably expressed regardless of the experimental conditions under study. We examined the expression variations of a set of eight candidate reference genes in tomato leaf and root tissues subjected to the infection of five taxonomically and molecularly different plant viruses and a viroid, inducing diverse pathogenic effects on inoculated plants. Parallel analyses by three commonly used dedicated algorithms, geNorm, NormFinder and BestKeeper, showed that different viral infections and tissues of origin influenced, to some extent, the expression levels of these genes. However, all algorithms showed high levels of stability for glyceraldehyde 3‐phosphate dehydrogenase and ubiquitin , indicated as the most suitable endogenous transcripts for normalization in both tissue types. Actin and uridylate kinase were also stably expressed throughout the infected tissues, whereas cyclophilin showed tissue‐specific expression stability only in root samples. By contrast, two widely employed reference genes, 18S ribosomal RNA and elongation factor 1α , demonstrated highly variable expression levels that should discourage their use for normalization. In addition, expression level analysis of ascorbate peroxidase and superoxide dismutase showed the modulation of the two genes in virus‐infected tomato leaves and roots. The relative quantification of the two genes varied according to the reference genes selected, thus highlighting the importance of the choice of the correct normalization method in such experiments.

Foodborne illnesses caused by the ingestion of foods contaminated with pathogens and/or their toxins are still one of the major public health threats worldwide. Disposable devices, allowing the on-site, early and multiplexed quantitative detection of pathogenic bacteria are therefore highly sought. Herein, we report biochips that are able to quantitatively detect two of the most common food-associated pathogens, namely Listeria monocytogenes and Staphylococcus aureus from the suspensions of bacteria stationary-phase broth culture. With a detection limit as low as 5.00 CFU ml⁻¹ for L. monocytogenes and 1.26 CFU ml⁻¹ for S. aureus, our platform may be a promising point-of-care device not only for clinical and food diagnostics but also for biosecurity purposes.

The population increase in the last century was the first cause of the industrialization of animal productions, together with the necessity to satisfy the high food demand and the lack of space and land for the husbandry practices. As a consequence, the farmers moved from extensive to intensive agricultural systems and introduced new practices, such as the administration of antimicrobial drugs. Antibiotics were then used as growth promoters and for disease prevention. The uncontrolled and continuous use of antibiotics contributed to the spread of antibiotic resistance in animals, and this had adverse impacts on human health. This emergence led the European Union, in 2003, to ban the marketing and use of antibiotics as growth promoters, and for prophylaxis purposes from January 2006. This ban caused problems in farms, due to the decrease in animal performances (weight gain, feed conversion ratio, reproduction, etc.), and the rise in the incidence of certain diseases, such as those induced by Clostridium perfringens, Salmonella, Escherichia coli, and Listeria monocytogenes. The economic losses due to the ban increased the interest in researching alternative strategies for the prophylaxis of infectious diseases and for health and growth promotion, such as feed additives. Yeast-based materials, such as cell wall extract, represent promising alternatives to antibiotics, on the base of their prebiotic activity and their claimed capacity to bind enteropathogenic bacteria. Several authors reported examples of the effectiveness of yeast cell wall products in adsorbing bacteria, but there is a lack of knowledge on the mechanisms involved in this interaction. The purpose of this review is to provide an overview of the current approaches used for the control of pathogenic bacteria in feed, with a particular focus on the use of yeast-derived materials proposed to control zoonoses at farm level, and on their effect on animal health.