**1. Introduction**

*Nosema ceranae* is a unicellular sporogenous fungus belonging to the phylum microsporidia, which gives rise to a chronic debilitating infection in honey bees named nosemosis [1]. This pathogen co-evolved with *Apis cerana*, whose parasitism became endemic in Asia. Nevertheless, *Apis mellifera* colonies infected by *N. ceranae* were found for the first time in 2005 in Taiwan [2] and in 2006 in most European countries [3]. When this host species shift occurred is unknown, although if it is reasonable to believe that it happened at the time of *A. mellifera* introduction in Asia, in the 1880s [4]. The exact *N. ceranae* arrival period in Europe is not clear but evidence suggests that it has been present in Europe since 1998 [5], thanks to an active international trading of *A. mellifera* from Asia to the rest of the world. Recently, a revision and redefinition of the genera Nosema and Vairimorpha proposes to rename *N. ceranae* and *N. apis* as *Vairimorpha ceranae* and *Vairimorpha apis* [6] which, more than taxonomic consequences, could become relevant for future research in

**Citation:** Braglia, C.; Alberoni, D.; Porrini, M.P.; Garrido, P.M.; Baffoni, L.; Di Gioia, D. Screening of Dietary Ingredients against the Honey Bee Parasite *Nosema ceranae*. *Pathogens* **2021**, *10*, 1117. https://doi.org/ 10.3390/pathogens10091117

Academic Editor: Giovanni Cilia

Received: 14 July 2021 Accepted: 27 August 2021 Published: 1 September 2021

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the topic. Adult honey bees easily become infected by ingesting spores from stored honey and pollen and subsequent transmission through trophallaxis [7,8], also after the exposure to surfaces contaminated by spores, following the colony cleaning and visits to contaminated flowers and pollen in the foraging activity [7,9]. After the ingestion, microsporidia spores germinate extracellularly in the midgut lumen and then inject the sporoplasm in an epithelial cell through the polar tube [1]. Infected colonies at the beginning show no visible symptoms and when environmental conditions are favorable for the parasite, they may rapidly collapse [10], making nosemosis a disease that sometimes is hard to control and difficult to diagnose and cure. The mycotoxin fumagillin (dicyclohexylamine salt), produced by *Aspergillus fumigatus*, is the first and the only successful antibiotic for the treatment of nosemosis [11–13] since 1953 [14]. Currently it is available on the market in many American countries and Korea, but it is forbidden in the European Union because of the absence of a detailed threshold residue regulation in honey and hive products [15]. Fumagillin use is nowadays controversial: targeting the methionine aminopeptidase-2 (MetAP2) [16], an enzyme present in many eukaryotes, can cause metabolic imbalances also in non-target organisms, i.e., it has been classified as mutagenic and cytotoxic for mammals after a short-term exposure [17]. Fumagillin residues persist in hives and its degradation products pose a potential risk for human health [18,19]. For instance, fumagillin toxicity was assessed in honey bees, causing a reduction in their lifespans [20], an alteration of structural and metabolic proteins in midgut [21], a reduction in sperm quality [22] and health [13]. Moreover, its efficacy depends on several factors such as seasonality and plantations [23]. Furthermore, in laboratory conditions, Huang et al., 2013 [21] have demonstrated that mature *N. ceranae* spore proliferation was similar in treated and untreated bees at the recommended fumagillin concentration (250 μg/L) and induced *N. ceranae* hyper proliferation when fumagillin concentration was 10 folds lower. Therefore, the need to find new strategies, which combine honey bee health protection and governmental standards in terms of food safety, gave a new pulse to the research of alternative solutions. Studies aimed at verifying the effect of veterinary drugs or commercial dietary supplements on the honey bee gut microbiota composition have been published recently. In recent years [24,25], researchers have begun to evaluate the use of plant extracts as *Nosema* control agents, such as thymol, oregano oil, carvacrol, cinnamaldehyde, resveratrol, and garlic based products, and treated bees showed lower *N. ceranae* infection rates compared to control [26–28]. Moreover, *Andrographis paniculate*, *Cryptocarya alba*, *Gevuina avellane*, *Artemisia dubia*, and *Laurus nobilis* extracts were tested with positive results [29–33], supporting a plant extract based strategy as a promising tool in the control of bee diseases. Brassicaceae seeds also showed promising results for their protective effects against N. ceranae spores at the laboratory level [34]. Furthermore, the use of beneficial microorganisms like *Bifidobacterium* and *Lactobacillus* strains [35,36], and *Bacillus subtilis* metabolites [37,38] showed encouraging results. For example, Baffoni et al. (2016) [35] demonstrated that probiotic treatment with *Lactobacillus* and *Bifidobacterium* strains reduced the presence of *Nosema* spores in naturally infected bees, thus proving the efficacy of a preventive microorganism-based strategy. Commercial probiotic preparation also based on lactic acid bacteria were found to be effective against the same parasite [27,36]. Similarly, De Piano et al. [39] demonstrated a strong relation between bacterial metabolites and the count of *N. ceranae* spores, showing a significant decrease after *Lactobacillus johnsonii* AJ5 administration. Additionally, organic acids produced by lactic acid bacteria present in the honey bee's environment (flowers, beebread, and gut), such as lactic acid, acetic acid, and phenyl-lactic acid, were tested, through feeding, against these microsporidia and showed a strong reduction in spore load in bees [38]. Therefore, these compounds have particular interest for the beekeeping sector. The aim of the present study was to test feed ingredients belonging to 4 different groups (organic acids, *Saccharomyces* and antibiotics, wine derivatives, and plants extracts) for their antimicrobial activity against *N. ceranae* in *A. mellifera* workers, but also to evaluate their toxicity on individual honey bees.
