DOI: 10.2478/JAS-2020-0030 J. APIC. SCI. VOL. 64 NO. 2 2020J. APIC. SCI. Vol. 64 No. 2 2020 Review paper PATHOGENESIS, EPIDEMIOLOGY AND VARIANTS OF MELISSOCOCCUS PLUTONIUS (EX WHITE), THE CAUSAL AGENT OF EUROPEAN FOULBROOD Adrián Ponce de León-Door1,2 Gerardo Pérez-Ordóñez2 Alejandro Romo-Chacón2 Claudio Rios-Velasco2 José D. J. Órnelas-Paz2 Paul B. Zamudio-Flores2 Carlos H. Acosta-Muñiz2* 1 Universidad Tecnologíca de la Babícora, Km. 1 S/N, Carretera Soto Maynez-Gómez Farías, C. P. 31963, Namiquipa, Chihuahua, Mexico 2 Centro de Investigación en Alimentación y Desarrollo A.C., Unidad Cuauhtémoc, Av. Río Conchos S/N, Parque Industrial. C. P. 31570, Apartado Postal 781, Cd. Cuauhté- moc, Chihuahua, Mexico *corresponding author: [email protected] Received: 17 January 2020; accepted: 27 August 2020 Abstract The bacterium Melissococcus plutonius is the etiologic agent of the European foulbrood (EFB), one of the most harmful bacterial diseases that causes the larvae of bees to have an intestinal infection. Although EFB has been known for more than a century and is practically present in all countries where beekeeping is practiced, the disease has been little studied compared to American foulbrood. Recently, great advances have been made to understand the disease and the interaction between the pathogen and its host. This review summarizes the research and advances to understand the disease. First, the morphological characteristics of M. plutonius, the infection process and bacterial development in the gut of the larva are described. Also, the epidemiological distribution of EFB and factors that favor the development of the disease as well as the classification of M. plutonius according its genomic and phenotype characteristics are reported. Finally, the new molecular tools for the study of M. plutonius, possible virulence factors in its genome, the issue of current EFB control measures and possible alternatives to the use of antibiotics are addressed. Keywords: Apis mellifera, European foulbrood disease, Melissococcus plutonius, pathogenesis, virulence INTRODUCTION most vital bacterial diseases of honey bees in the world is European Foulbrood (EFB) caused Honey bees (mainly Apis mellifera) are the most by the bacterium Melissococcus plutonius. EFB valuable pollinators in the world. They are inex- is generally associated with such secondary pensive, versatile, and usually, the only solution bacteria as Enterococcus faecalis, Brevibacillus to guarantee pollination (Morse & Calderone, laterosporus, Bacillus pumilis, Paenibacillus alvei, 2000). However, their health is impacted by and Paenibacillus dendritiformis that may lead such numerous pathogens as bacteria, viruses, to a pathogenic effect, but their role in the de- fungi and parasites. The factors involved in velopment of the disease is unclear (Forsgren, bee’s health affect sustainable and profitable et al., 2018). M. plutonius leads to intestinal agriculture, as well as many non-agricultur- infection in bee larvae causing death between al ecosystems, (Genersch, 2010). One of the the fourth and seventh days. Infected larvae 173 de León-Door et AL. Pathogenesis of Melissococcus plutonius have an abnormal position inside the brood cell cal and chemical characteristics. The specific and the color of the larvae changes from pearly epithet ‘pluton’ was corrected to plutonius by white to yellow, then brown and finally, when Dicks, Endo, & Van Reenen (2014) and Trüper & they decompose to grayish-black (Bailey, 1961). de’Clari (1998). M. plutonius is a Gram-positive Occasionally, the larvae die after the brood cocci lanceolate oval-shaped, non-spore-forming cell is sealed. Present P. alvei remains and bacteria, with an approximate size of 0.5 x 1.0 multiplies in the larvae and sunken opercula can μm (Fig. 1), that is found individually, in pairs be observed, and these symptoms are possibly or chains of various lengths (Forsgren, 2010; confused with American foulbrood (Bailey & Forsgren et al., 2013). The bacteria only affect Ball, 1991; Forsgren, 2010). M. plutonius infects the larval stages of bees (Bailey & Collins, 1982). European bees A. mellifera, Asian bees A. cerana The infection begins with the consumption of (Singh Rana et al., 2012; Takamatsu et al., 2014) contaminated food during the early larval stage, and Himalayan bees A. laboriosa (Allen, Ball, & when the larvae are exclusively fed with royal Underwood, 1990). The etiologic agent of EFB jelly (RJ), a mixture of secretions from the hy- was identified more than a century ago (White, popharyngeal and mandibular glands of young 1912). To understand and treat a disease, the worker bees (Snodgrass, 1925). causative agent, mechanisms of infection, and Royal jelly (RJ) has high antimicrobial activity favorable conditions for its development must (Melliou & Chinou, 2014) due to proteins like be identified. This review summarizes recent major royal jelly protein 1 (MRJP1) containing research on the epidemiology and pathogenesis jelleins 1, 2 and 3, which inhibit bacterial, yeast of M. plutonius, the causative agent of EFB. and fungus growth (Brudzynski & Sjaarda, 2015; Fontana et al., 2004). Recently Vezeteu Pathogenesis and Epidemiology et al. (2017), showed how MRJP1 can inhibit the Melissococcus plutonius was first classified development of bacteria associated with EFB, as Bacillus pluton (White, 1912) and later as including M. plutonius. MRJP2 has an antibacterial Streptococcus pluton (Bailey, 1957b). However, effect against P. larvae (Bíliková, Wu, & Šimúth, Bailey, & Collins (1982) described Melissococcus 2001) and the enzyme glucose oxidase, which is as a new genus, containing the species Melis- essential to inhibiting microbial development in sococcus pluton based on culture, biochemi- larval foods and honey (Ohashi, Natori, & Kubo, Fig. 1. Gram staining of Melissococcus plutonius strain ATCC 35311, Cultivated under anaerobic conditions in SBK medium for 5 days at 36°C. 174 J. APIC. SCI. Vol. 64 No. 2 2020 1999; Sano et al., 2004). On the other hand, such the death of the larvae may be the result of non-protein components as 10-hydroxy-2-dece- such additional pathogenic mechanisms as peri- noic acid (10-HDA) have demonstrated antipath- trophic matrix invasion and penetration into ogenic activity. Šedivá et al. (2018) showed that other host tissues. However, Takamatsu, Sato, & 10-HDA contributes to the inhibition of P. larvae, Yoshiyama (2016) refuted this theory inferring and Yang, Li, & Wang (2015) proposed that it is a that the presence of substances produced by broad-spectrum antimicrobial agent that inhibits M. plutonius can diffuse into larval tissues during multiple pathogenic bacteria. However, despite the infection, but these substances and other the RJ antimicrobial activity, M. plutonius can virulence factors remains unidentified. Only 100 survive and infect honey bee larvae through it to 200 bacteria cells are required to cause EFB (Takamatsu et al., 2017). (Bailey, 1960; McKee et al., 2004), and once the Studies show that after ingestion, M. plutonius infection is established, the larvae can die before reaches the middle intestine of the larvae, the brood cell is covered. In this case, larvae are where it begins to multiply until it almost expelled from the colony or may die after the occupies the intestinal lumen without crossing brood cell is covered where the sunken upper the peritrophic membrane (Tarr, 1938). White wax layer is observed (Fig. 2-5c). Sometimes, (1912) determined that M. plutonius grows only the larvae survive the infection, however, in the food mass within the peritrophic matrix, pupation is delayed (McKee et al., 2004), and killing its host before any bacteria associated smaller adults emerge (Bailey, 1959b), which with EFB succeeds in invading the larval tissues. can transmit the bacteria (Fig. 2-6b). Derived from this, Bailey (1983) suggested that Melissococcus plutonius multiplies only within the pathogenic effect was due to nutrient com- the larval intestine of the honey bee. The per- petition between the larva and the pathogen, sistence of the pathogen in the hive depends resulting in starvation of the larvae. McKee, on the infected larvae’s survival, which Goodman, & Hornitzky (2004) suggested that deposits the bacteria along with their feces Fig. 2. Melissococcus plutonius infection cycle 1. egg, 2. Ingestion of M. plutonius (Mp) in the royal jelly (RJ). 3. Proliferation of M. plutonius in the intestine, 4a. Manifestation of EFB symptoms, 4b Larvae without EFB symptoms, 5a. Death of larvae, scale formation and brood removal by worker bees, 5b. Sealing cell and slow pupae development, 5c. Death of larva after cell was seal, 6a. Cell with M. plutonius and secondary agents, 6b. Emergence of smaller adults carrying M. plutonius and cells with M. plutonius and secondary agents. Adapted from Bailey & Ball (1991). 175 de León-Door et AL. Pathogenesis of Melissococcus plutonius in the brood cell when they pupate (Fig. 2-6a, apiaries (Belloy et al., 2007). In Switzerland, a 6b). M. plutonius stay viable in the brood cell, high density of colonies and hives have been surviving for several years (Bailey, 1959a). A shown to promote the transmission of EFB large number of bacterial cells die during this (von Büren et al., 2019). Additionally, Abrol time, but the remaining bacteria can infect (2013) and Forsgren (2010) suggested that the other larvae. If the infected larva dies before it robbing of honey and drifting bees contributed becomes a pupa, the worker bees eliminate the to the spread of the bacteria between colonies infected larvae (Fig. 2-5a), reducing the number and apiaries, but in contrast Goodwin, Perry, & of bacteria that serve as a source of inoculum. Houten (1994) suggested that the drift of bees is Nevertheless, worker bees will subsequently not an important to the spread of AFB. However, feed new larvae causing the transmission of the it is not clear how EFB spreads reapidly, but such bacteria (Fig. 2-2). The infection is not always beekeeping practices as the exchange of con- fatal, and M. plutonius can be present in larvae taminated combs, poor equipment sanitation and pupae without any clinical symptoms (Fig. 2- and poor diet may contribute to it (Fig.