**1. Introduction**

*Aethina tumida* (Murray 1867), the small hive beetle (SHB), is a coleopteran species belonging to the Nitidulidae family [1]. Native to Sub-Saharan Africa [2], it is a destructive, invasive pest of *Apis mellifera* (western honey bee) colonies [3], and it causes significant damage to brood, pollen, and honey stores [4]. Presently, the SHB is recorded in all continents except Antarctica [3,5–8], having reached North America in 1996; Australia in 2000; and, more recently, countries in Europe, South America, and Asia [9–12]. The SHB is an ecological generalist [4] and creates persistent populations in colonies in areas in which it has been introduced [13].

Honey bees are exposed to pests and pathogens belonging to different groups (viruses, bacteria, fungi, protists, mites, insects, etc.), some of which are responsible for severe health impairment and colony collapse [14–17]. Adult SHBs invade colonies, where they feed, thrive, and reproduce. This allows contact between SHBs and other bee pests and pathogens [18–23].

*Lotmaria passim* and *Crithidia mellificae* are two trypanosomatid species capable of colonizing the digestive system of honey bees [24,25]. The transmission is deemed to occur by the oral–fecal route [26,27], and the presence of infected faeces within the hive may promote the circulation of the parasite among worker bees [26]. Both pathogens are deemed to impact colony health by altering bee behavior, physiology, immune response, and lifespan [28–31]. Nevertheless, the details of their pathogenic effects are still not fully understood. *Lotmaria passim* has been described only recently [25], and it is presently

**Citation:** Nanetti, A.; Ellis, J.D.; Cardaio, I.; Cilia, G. Detection of *Lotmaria passim*, *Crithidia mellificae* and Replicative Forms of Deformed Wing Virus and Kashmir Bee Virus in the Small Hive Beetle (*Aethina tumida*). *Pathogens* **2021**, *10*, 372. https:// doi.org/10.3390/pathogens10030372

Academic Editor: Lawrence S. Young

Received: 16 February 2021 Accepted: 18 March 2021 Published: 19 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

acknowledged as the most prevalent *A. mellifera* trypanosomatid [32]. Infections have been reported in Asian, European, and South and North American colonies [8], whereas *C. mellificae* infections have been rarely observed [8,33–35].

Deformed wing virus (DWV) is a positive-sense ssRNA virus belonging to the Picornaviridae family within the Iflavirus genus [36,37]. Spread globally [32,36–38], three genetic variants have been acknowledged and identified as types A, B, and C [39,40]. Type A is by far the most widespread [40], and it may generate asymptomatic or symptomatic infections, the latter including deformed or missing wings, shortened abdomens, and premature bee death [36]. Generally, this virus is transmitted through puncture wounds produced by the ectoparasite *Varroa destructor* as it feeds on immature honey bees [41]. However, the infection may be transmitted horizontally by bee-to-bee contact, especially in cases of severe infections [42–46], curbicular pollen, bee products, and floral contamination [47–49].

Kashmir bee virus (KBV) is a positive-sense ssRNA virus of the Dicistroviridae family within the Cripavirus genus [50,51], considered endemic in North America and Australia [52,53] but rarely reported in Europe [54–58]. It is genetically related to acute bee paralysis virus (ABPV) [59], and the two may co-infect the same colony or the same individual bee [59,60]. Low viral titers are generally detected in subclinical colonies; however, viral replication may be triggered by the presence of stressors, including *A. tumida* infestations [46,52,60], with a lethal outcome for different honey bee stages [59,61,62]. Ingestion of contaminated brood food [49,59,63] and *Varroa* feeding behavior [64–66] may elicit the transmission of KBV infections.

Herein, we aimed to assess the presence of the abovementioned pathogens (*L. passim, C. mellificae*, KBV, and DWV) in addition to pathogenic bacteria (*Paenibacillus larvae* and *Melissococcus plutonius*) and other bee viruses (ABPV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), sacbrood virus (SBV), chronic bee paralysis virus (CBPV), and slow paralysis virus (SPV, major and minor)) in SHB specimens collected in Florida, USA in 2017. This is an important first step in determining the role SHBs may plan in the movement of pathogens between honey bee colonies.
