Atypical Melissococcus plutonius strains: their characteristics, virulence, epidemiology, and mysteries

Abstract

Melissococcus plutonius is a Gram-positive lanceolate coccus that is the causative agent of European foulbrood, an important bacterial disease of honey bee brood. Although this bacterium was originally described in the early 20th century, a culture method for this bacterium was not established until more than 40 years after its discovery due to its fastidious characteristics, including the requirement for high potassium and anaerobic/microaerophilic conditions. These characteristics were considered to be common to the majority of M. plutonius strains isolated worldwide, and M. plutonius was also thought to be genetically homologous or clonal for years. However, non-fastidious variants of this species (designated as atypical M. plutonius) were very recently identified in Japan. Although the morphology of these unusual strains was similar to that of traditionally well-known M. plutonius strains, atypical strains were genetically very different from most of the M. plutonius strains previously isolated and were highly virulent to individual bee larva. These atypical variants were initially considered to be unique to Japan, but were subsequently found worldwide; however, the frequency of isolation varied from country to country. The background of the discovery of atypical M. plutonius in Japan and current knowledge on atypical strains, including their biochemical and culture characteristics, virulence, detection methods, and global distribution, are described in this review. Remaining mysteries related to atypical M. plutonius and directions for future research are also discussed.

Pollination by insects is an essential component of agricultural and horticultural crop production. Honey bees, particularly Apis mellifera, are one of the most important commercial pollinators that support agriculture worldwide. In Japan, as of January 1, 2022, 242,000 honey bee colonies were being kept by 11,276 farm households (https://www.maff.go.jp/j/chikusan/kikaku/lin/sonota/attach/pdf/bee-45.pdf [accessed on June 12th, 2023]), and bees are also used in this country to pollinate various fruits and vegetables. Therefore, the maintenance of healthy bee colonies is important not only for honey production, but also for stable food production and the rich dietary life of humans. The healthy development of larvae is vital for maintaining healthy colonies because most larvae grow into worker bees, which are responsible for all the tasks needed to maintain the colony other than egg-laying, such as the cleaning and construction of hives, rearing larvae, colony defenses, foraging, and food storage. Therefore, infectious diseases and parasites that affect honey bee larvae seriously threaten the maintenance of healthy bee colonies.

American foulbrood (AFB) and European foulbrood (EFB) are two major bacterial infectious diseases affecting bee larvae [23, 28]. Both diseases have been reported in most countries and regions where beekeeping is practiced [18]. Outbreaks of these diseases may cause colony collapse and result in economic losses in the agricultural industry; therefore, they are recognized as important honey bee diseases and are listed in the World Organization for Animal Health Terrestrial Animal Health Code (https://www.woah.org/en/disease/diseases-of-bees/ [accessed on June 12th, 2023]). In Japan, foulbroods are also important in beekeeping and designated as “monitored infectious diseases” under the Act on the Prevention of Infectious Diseases in Livestock (Act No. 166 of 1951), and every year, a hundred or more honey bee colonies are diagnosed with foulbrood diseases and reported to the government. However, despite their importance, our understanding of foulbrood, particularly EFB, is still very limited.

EFB mainly affects unsealed larvae and typically kills them at 4–5 days old. Larvae that die from the infection cover the bottom of their cells or become malpositioned and flaccid in their cells; twisted around the walls or stretched out lengthways. Dead larvae change from pearly white to yellow and then brown, decompose, and finally become grayish black. Some larvae may also die after the cell is capped, resulting in sunken capping resembling the symptoms of AFB. In severely affected colonies, the brood pattern appears patchy, with capped and uncapped cells being irregularly scattered over the brood frame and often giving off a foul or sour smell due to the decomposition of larval remains by secondary invaders, such as Enterococcus faecalis and Paenibacillus alvei [4, 23, 24].

The causative agent of EFB is the Gram-positive lanceolate coccus, Melissococcus plutonius. This bacterium was originally described in 1912 by White as Bacillus pluton [74] and was classified into the genus Streptococcus as Streptococcus pluton in Bailey’s study in 1957 [6]. In 1982, it was reclassified by Bailey and Collins as the only species in the new genus, Melissococcus pluton, based on culture, biochemical and chemical characteristics [10], and in 1998, the species name was corrected from pluton to plutonius [72]. Although this bacterium was discovered more than 100 years ago [74], its cultivation was extremely difficult due to its fastidious characteristics. After the efforts of our predecessors, Bailey finally found and reported in 1957 that a high inorganic phosphate concentration, glucose or fructose, and factors from yeast extract are required for the anaerobic growth of this bacterium [6]. Furthermore, the Na:K ratio required for growth is described to be 1 or less in the report [6]; that is, low sodium and high potassium conditions are necessary for the growth. Therefore, the addition of potassium phosphate to culture media is required for the isolation and maintenance of this bacterium. Although a few unusual isolates that appear to be M. plutonius were reported [8, 9], isolates of this species were considered to be homologous or clonal based on morphological, physiological, immunological, and genetic studies [1, 11, 16, 23]; therefore, these fastidious characteristics were considered to be common to strains of M. plutonius.

However, M. plutonius strains that do not fit the previously reported characteristics of this bacterium were identified in Japan. Although these strains (designated as atypical strains) were initially considered to be unique to Japan, it became increasingly clear that they exist worldwide and may be dominant in some areas, and, thus, have been attracting increasing attention in recent years. In this review, current knowledge on atypical strains is described, and remaining mysteries related to atypical M. plutonius and future perspectives are discussed.

DISCOVERY OF ATYPICAL M. PLUTONIUS

In Japan, the Ministry of Agriculture, Forestry, and Fisheries has compiled standard methods for pathological appraisals of livestock diseases and presented them as “the guidelines for pathological appraisals” to promote infectious disease control in livestock. The guidelines describe test methods for EFB, and similar to Bailey’s statement [6, 10], the third edition of the guidelines, which were in use until it was revised in 2015, stated that M. plutonius only grows at a Na:K ratio less than 1 and its growth is negative on Brain Heart Infusion (BHI) agar, but positive on BHI agar supplemented with 0.15 M KH₂PO₄. Although the presence of EFB in Japan was confirmed in the 1980s [78], the characteristics of the causative strains initially isolated were consistent with those described in the guidelines and the study by Bailey [6], and the addition of potassium phosphate to the medium was necessary for their growth. However, in the 1990s, Tominaga isolated M. plutonius-like organisms from diseased honey bee larvae that differed from typical M. plutonius characteristics, and similar organisms were rediscovered in the 2000s by Arai et al [4]. These M. plutonius-like isolates were morphologically similar to M. plutonius (Fig. 1) and positive for M. plutonius-specific PCR targeting the 16S rRNA gene [32]. However, they were able to grow on medium supplemented with sodium phosphate (i.e., independently of the Na:K ratio) [4]. Therefore, to establish their taxonomic position, Arai et al. [4] performed 16S rRNA gene sequencing and DNA-DNA hybridization analyses and showed that their 16S rRNA sequences were more than 99.8% homologous to that of the type strain of M. plutonius and also that the levels of genome DNA relatedness between the type strain and M. plutonius-like isolates were higher than 80%. Since values of 98.65% and 70% were proposed as the recommended standards for delineating species in 16S rRNA gene sequencing and DNA-DNA hybridization analyses, respectively [39, 73], these findings confirmed that M. plutonius-like organisms were taxonomically identical to M. plutonius. The average nucleotide identity values calculated from the recently obtained whole genome sequence data of representative M. plutonius strains [50,51,52] also support this conclusion (Supplementary Table 1). However, by a macrorestriction analysis of SmaI-digested genomic DNA using pulsed-field gel electrophoresis, these M. plutonius-like isolates were classified into a cluster that was genetically distinct from typical M. plutonius isolates [4]. Therefore, the M. plutonius-like organisms discovered in Japan were designated as atypical M. plutonius in the study by Arai et al. [4].

Fig. 1.

Colony and bacterial cell morphologies of representative typical and atypical Melissococcus plutonius strains (DAT606 and DAT1179, respectively) cultured on KSBHI agar [4] at 35°C for five days under anaerobic conditions.

Before the discovery of atypical M. plutonius, several putative M. plutonius strains, which grew weakly or moderately even on media supplemented with sodium phosphate, had been isolated from samples collected in Brazil [1, 9]. Although BHI was not used in these studies, the non-fastidious culture characteristics of the Brazilian strains (i.e., the growth ability under high sodium and low potassium conditions) were similar to those of the Japanese atypical strains. Since the Brazilian strains were not analyzed by molecular approaches such as DNA-DNA hybridization and pulsed-field gel electrophoresis analyses, at least in these studies [1, 9], their taxonomic positions and relationships to Japanese atypical isolates are unclear. However, their unusual characteristics imply that the Brazilian strains are also atypical M. plutonius.

PHENOTYPIC DIFFERENCES BETWEEN ATYPICAL AND TYPICAL STRAINS

Major commonalities and differences in culture and biochemical characteristics between typical and atypical M. plutonius are summarized in Table 1. As described by Bailey [6], typical M. plutonius strains are fastidious in their culture requirements. On media supplemented with potassium phosphate (e.g., KSBHI and Medium 1), they grew well under anaerobic conditions (Table 1) [4]. However, under high sodium and low potassium conditions (e.g., BHI and Medium 6) or under aerobic conditions, their growth was completely inhibited (Table 1) [4]. In contrast, the culture requirements of atypical M. plutonius strains are not fastidious. On low-sodium-and-high-potassium media (e.g., KSBHI and Medium 1), they grew not only anaerobically, but also aerobically (Table 1) [4]. Moreover, under anaerobic conditions, atypical strains grew even on media not supplemented with potassium phosphate (e.g., BHI and Medium 6) (Table 1) [4].

Table 1. Major commonalities and differences in culture and biochemical characteristics between typical and atypical Melissococcus plutonius^a.

Conditions or tests			Results for
			Typical	Atypical
	Growth onb	Condition
Commonality	KSBHI agar (K >Na)	Anaerobic	+	+
	BHI agar (Na >K)	Aerobic	-	-
	Medium 1 (K >Na)	Anaerobic	+	+
	Medium 6 (Na >K)	Aerobic	-	-
Difference	KSBHI agar (K >Na)	Aerobic	-	+
	BHI agar (Na >K)	Anaerobic	-	+
	Medium 1 (K >Na)	Aerobic	-	+
	Medium 6 (Na >K)	Anaerobic	-	+
	Enzyme activity
Commonality	Catalase		-	-
	Oxidase		-	-
	Nitrate reduction		-	-
	Production of indole		-	-
	Urease		-	-
Difference	Hydrolysis of esculin (β-glucosidase)		-	+
	Production of acids from carbohydrates
Commonality	Glucose		+	+
	Fructose		+	+
	D-Mannose		+	+
Difference	L-Arabinose		-	+
	D-Cellobiose		-	+c
	Salicin		-	+c

^a Data were retrieved from the previous study by Arai et al. [4]. ^b KSBHI agar (37 g/L brain heart infusion [BHI], 10 g/L soluble starch, 0.15 M KH₂PO₄, 15 g/L agar); BHI agar (37 g/L brain heart infusion, 15 g/L agar); Medium 1 (10 g/L yeast extract, 10 g/L glucose, 10 g/L soluble starch, 0.1 M KH₂PO₄, 15 g/L agar [pH 6.6]); Medium 6 (10 g/L yeast extract, 10 g/L glucose, 10 g/L soluble starch, 0.1 M NaH₂PO₄•2H₂O, 15 g/L agar [pH 6.6]). ^c Some atypical strains showed only very weak acid production.

Potassium is a major intracellular cation [19], and the intracellular accumulation of K⁺ is known to play a primary role in maintaining the osmotic balance of the cell in some bacteria [60]. To make room for intracellular K⁺ accumulation, the elimination of Na⁺ from the cytoplasm by sodium extrusion systems is speculated to be important [38]. However, in typical M. plutonius, a putative Na⁺/H⁺ antiporter gene (napA) and a cation-transporting ATPase gene (ctaP), which are considered to be involved in sodium extrusion, were found to be mutated [61]; therefore, typical strains may have lost the ability to eliminate Na⁺ from the cytoplasm and thus, require the addition of potassium salts to culture media in order to make the environmental K⁺ concentration high and take K⁺ in efficiently by osmotic pressure. On the other hand, atypical M. plutonius has one of the putative sodium extrusion system genes (ctaP), and the presence of an intact ctaP gene is considered to be the reason why atypical strains do not require the addition of potassium for the growth [61].

Some biochemical characteristics also differ between the two types. Although all typical M. plutonius strains tested in a previous study [4] produced acid from glucose, fructose, and D-mannose, they did not utilize the other carbohydrates tested. In contrast, in addition to the three sugars, atypical strains produced acids from L-arabinose, D-cellobiose, and salicin [4], although some strains only showed very weak acid production from D-cellobiose and salicin (Takamatsu D., unpublished observations). Moreover, only atypical strains, but not typical strains, can hydrolyze esculin. The utilization of arabinose and salicin was also reported in the non-fastidious Brazilian M. plutonius strains [1]. These findings further suggest that the Brazilian strains are atypical.

In addition to culture and biochemical characteristics, the antigenicity of the bacterial cell surface appears to differ between typical and atypical strains. For example, although typical and atypical strains are detectable by the commercially available lateral flow device, EFB Diagnostic Test Kit (https://www.vita-europe.com/beehealth/products/efb-diagnostic-test-kit/ [accessed on June 12th, 2023]), which was designed for the detection of M. plutonius using specific monoclonal antibodies [70], detection sensitivity was lower for atypical strains [63]. Similarly, the putative atypical strains isolated in Brazil poorly reacted to antisera prepared against putative typical strains isolated in the UK and the USA [1, 9]. These findings suggest different cell surface structures between typical and atypical strains.

The efficacy of chlorine-based disinfectants (slightly acidic hypochlorous acid water and weakly acidified chlorous acid water [WACAW]) also varies between atypical and typical strains. In suspension tests, the representative atypical strain was more resistant to these disinfectants than representative typical strains [48]. Although the antimicrobial activity of chlorine-based disinfectants generally decreases in the presence of organic matter, WACAW was shown to be relatively stable under organic matter-rich conditions [31, 37]. WACAW with an available chlorine concentration of 2,400 ppm reduced viable typical M. plutonius cells (1.93– >5 log₁₀ CFU/mL reduction after a 24-hr treatment) even under the 1% yeast extract plus 1% bovine serum albumin conditions. However, the death of the atypical strain tested under the same conditions even by the WACAW was negligible (only ≤0.55 log₁₀ CFU/mL reduction after a 24-hr treatment) [48]. The microbicidal effects of chlorine-based disinfectants are attributed to their oxidative potential. As described above, atypical strains, but not typical strains, can grow even under aerobic conditions [4], implying that atypical strains are more resistant to oxidative stress than typical strains. This difference in oxidative stress susceptibility may result in the different disinfectant susceptibilities between the two types [48].

POPULATION STRUCTURE OF INTERNATIONAL M. PLUTONIUS ISOLATES AND PHYLOGENETIC RELATIONSHIPS BETWEEN ATYPICAL AND TYPICAL STRAINS

In 2013, Haynes et al. published the multilocus sequence typing (MLST) scheme developed for the genotyping of M. plutonius isolates using the sequences of four loci on the genome [36]. This scheme has since been used in molecular epidemiological analyses of M. plutonius isolates [13, 14, 35, 65, 69], and most of the typing results obtained to date have been deposited in the MLST database (https://pubmlst.org/organisms/melissococcus-plutonius [accessed on June 12th, 2023]). As mentioned above, atypical M. plutonius isolates were classified into a cluster genetically distinct from typical M. plutonius isolates by a pulsed-field gel electrophoresis analysis [4]. When the population structure of M. plutonius is analyzed by the goeBURST algorithm on the PHYLOViZ Online site (https://online.phyloviz.net/index [accessed on June 12th, 2023]) using MLST data deposited in the database and reported in previous studies [14, 76], international M. plutonius isolates assigned to 40 sequence types can be grouped into three clonal complexes (CC3, CC12, and CC13) (Fig. 2). All typical strains have been assigned to CC3 or CC13, which are two groups that are relatively close to each other. In contrast, all atypical strains were assigned to CC12, which was distantly related to the two other groups (Fig. 2) [65]. These findings again suggested a distant relationship between typical and atypical strains, and a genome analysis [17] of M. plutonius isolates also supported this relationship. Although atypical strains were initially considered to be unique to Japan, MLST analyses revealed the worldwide distribution of atypical M. plutonius because CC12 also included international strains from Europe and North and South America (Fig. 2) [13, 14, 35, 65, 69]. Therefore, it remains unclear when and from where these atypical strains originated. However, some findings from genomic and phylogenetic analyses have provided some insight into the origin of atypical strains. An investigation on the evolutionary history of several housekeeping genes of M. plutonius isolates and an E. faecalis strain (as an outgroup) suggested that typical and atypical types had a common ancestor and had branched off from the ancestor a long time ago (Fig. 3). In addition, genome analyses of representative strains revealed the presence of many (more than 160) pseudogenes (i.e., mutated non-functional genes) in the genomes of typical (CC3 and CC13) strains [51, 52]. In contrast, the number of pseudogenes in the representative atypical (CC12) strain was only 21 [51, 52], and many genes that were not functional in typical strains were predicted to be functional in the atypical strain. One example is the ctaP gene described in Phenotypic differences between atypical and typical strains, which allows the growth of atypical strains under high sodium conditions [61]. Another example is the rlmA^II gene that encodes the rRNA large subunit methyltransferase and confers specific resistance to mirosamicin but has little impact on the susceptibility of M. plutonius to other macrolides, including tylosin [67]. Regardless of the genotype, all M. plutonius strains examined in the previous study had this gene on their chromosome; however, many typical strains had lost its function due to truncation by a single-nucleotide insertion and, thus, lost their resistance to mirosamicin [67]. In contrast, all atypical strains tested had the functional rlmA^II gene [67]. These findings suggest that atypical strains retain many of their ancestral characteristics that typical strains have lost, i.e., atypical M. plutonius is not a variant that has recently diverged from typical M. plutonius but a type that has existed for a long time.

Fig. 2.

The goeBURST tree of sequence types (STs) found in Melissococcus plutonius isolates from different countries. Data available in the M. plutonius multilocus sequence typing database (https://pubmlst.org/organisms/melissococcus-plutonius) as of February 28, 2023 and data on Mexican strains reported by de León-Door et al. [14] and a Canadian strain reported by Wood et al. [76] were used to construct the tree. Each circle represents a different ST, with lines linking closest relatives. Black lines indicate a single allelic change between STs. Light gray lines indicate differences at two loci. Circles ringed with a green outline indicate putative founder genotypes. Colors within circles show the percentage of isolates of a particular type that were found in the countries indicated.

Fig. 3.

The evolutionary history of Melissococcus plutonius gyrA (A) and recN (B) genes inferred using the neighbor-joining method [58]. Nucleotide sequences of the genes of each M. plutonius strain were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/nucleotide/ [accessed on June 12th, 2023]). The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) [21] are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed using the p-distance method [46] and are shown in the units of the number of base differences per site. All positions containing gaps and missing data were eliminated (complete deletion option). Evolutionary analyses were conducted in MEGA11 [68].

VIRULENCE OF ATYPICAL M. PLUTONIUS

A. mellifera larvae may be reared to adult bees in vitro (i.e., in laboratories) using artificial diets and 48-well plastic plates, and the virulence of M. plutonius strains at the individual insect level may be assessed by feeding larval food contaminated with the strains of interest under these rearing conditions. This assay method is called an exposure bioassay and has been performed in many laboratories conducting EFB research. The representative findings of exposure bioassays conducted to date are summarized in Table 2.

Table 2. Summary of representative findings of exposure bioassays using the in vitro rearing of Apis mellifera larvae^a.

Reference	Strain (typical/atypical)	Genotypeb		pMP19 possession at the time of the experimentc	Country of origin	Age of larvae at the start of the inoculation	Infection dosed	Observation period	Mortality of larvae at the end of the experiment
		ST	CC
[4]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	5 days	8.6%
	DAT606 (typical)	3	3	Probably −	Japan	<24 hr old	approx. 5.0 × 104 CFU/larva	5 days	5.7%
	DAT583 (typical)	3	3	Probably −	Japan	<24 hr old	approx. 5.0 × 104 CFU/larva	5 days	12.5%
	DAT585 (typical)	26	13	−	Japan	<24 hr old	approx. 5.0 × 104 CFU/larva	5 days	16.7%
	DAT561 (atypical)	12	12	Probably −	Japan	<24 hr old	approx. 5.0 × 104 CFU/larva	5 days	94.3%
	DAT351 (atypical)	12	12	−	Japan	<24 hr old	approx. 5.0 × 104 CFU/larva	5 days	91.7%
	DAT573 (atypical)	12	12	Probably −	Japan	<24 hr old	approx. 5.0 × 104 CFU/larva	5 days	70.8%
[77]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	5 days	0%
	DAT561 (atypical)	12	12	NI	Japan	<24 hr old	approx. 1.0 × 105 CFU/larva	5 days	83.3%
[45]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	6 days	2.9–11.1%
								21 days	8.6%
	DAT606 (typical)	3	3	Probably +	Japan	<24 hr old	6.0 × 103 CFU/larva	6 days	63.3%
							5.7 × 103 CFU/larva	21 days	100%
	DAT585 (typical)	26	13	−	Japan	<24 hr old	3.6 × 104 CFU/larva	6 days	7.1%
							2.5 × 103 CFU/larva	21 days	17.1%
	DAT561 (atypical)	12	12	Probably +	Japan	<24 hr old	6.7 × 103 CFU/larva	6 days	96.7%
							2.8 × 103 CFU/larva	21 days	100%
[43]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	21 days	5.3–19.4%
	DAT606 (typical)	3	3	+	Japan	approx. 48–72 hr old	6.6 × 102 −9.0 × 104 CFU/larva	21 days	55.3–97.5%
				−			2.0 × 102 −1.4 × 105 CFU/larva		0–35.0%
	DAT639 (typical)	3	3	+	Japan	approx. 48–72 hr old	7.6 × 103 CFU/larva	21 days	85.7%
				−			1.52 × 104 CFU/larva		5.6%
	DAT869 (typical)	4	13	+	Japan	approx. 48–72 hr old	8.0 × 103 CFU/larva	21 days	10.0%
				−			1.28 × 105 CFU/larva		10.0%
	DAT561 (atypical)	12	12	+	Japan	approx. 48–72 hr old	1.92 × 105 CFU/larva	21 days	100%
				−			1.94 × 105 CFU/larva		87.5%
[44]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	21 days	≤14.6%
	DAT561 (atypical)	12	12	−	Japan	approx. 48–72 hr old	2.2–4.6 × 104 CFU/larva	21 days	77.1–80.6%
[55]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	14 days	approx. 15%e
	119 (typical)	20	13	−	Switzerland	5–10 hr old	~6.0 × 104 CFU/larva	14 days	approx. 67%e
[40]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	12 days	approx. 24–41%?f
	49.3 (typical)	3	3	Probably +	Switzerland	<24 hr old	1.21 × 103 ± 398 CFU/larva	12 days	approx. 89–98%f
							3.46 × 104 ± 1.57 × 104 CFU/larva	14 days	approx. 50%f
	119 (typical)	20	13	−	Switzerland	<24 hr old	797 ± 380 CFU/larva	12 days	approx. 58–64%?f
	4–127 (typical)	1	13	NI	Sweden	<24 hr old	1.75 × 104 ± 8.42 × 103 CFU/larva	12 days	approx. 58–80%f
[34]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	21 days	17.98–21.25%
	UK 31.1 (typical)	13	13	−	England	<24 hr old	1 × 105 CFU/larva	21 days	4.67%g
	CH 82 (typical)	32	13	−	Switzerland	<24 hr old	1 × 105 CFU/larva	21 days	6.80%g
	FR 27.1 (typical)	20	13	−	France	<24 hr old	1 × 105 CFU/larva	21 days	16.46%g
	CH 90 (typical)	13	13	−	Switzerland	<24 hr old	1 × 105 CFU/larva	21 days	22.55%g
	CH 119 (typical)	20	13	−	Switzerland	<24 hr old	1 × 105 CFU/larva	21 days	28.28%g
	CH 54.1 (typical)	35	13	−	Switzerland	<24 hr old	1 × 105 CFU/larva	21 days	32.82%g
	CH 40.2 (typical)	35	13	−	Switzerland	<24 hr old	1 × 105 CFU/larva	21 days	43.97%g
	UK 36.1 (typical)	3	3	−	England	<24 hr old	1 × 105 CFU/larva	21 days	1.24%g
	IT 1.3 (typical)	3	3	−	Italy	<24 hr old	1 × 105 CFU/larva	21 days	7.43%g
	CH 46.1 (typical)	7	3	−	Switzerland	<24 hr old	1 × 105 CFU/larva	21 days	39.44%g
	CH 45.1 (typical)	3	3	−	Switzerland	<24 hr old	1 × 105 CFU/larva	21 days	42.93%g
	NO 764-5B (typical)	3	3	−	Norway	<24 hr old	1 × 105 CFU/larva	21 days	48.74%g
	NO 765-6B (typical)	3	3	−	Norway	<24 hr old	1 × 105 CFU/larva	21 days	53.50%g
	CH 49.3 (typical)	3	3	+	Switzerland	<24 hr old	1 × 105 CFU/larva	21 days	79.98%g
				−					5.88%g
	CH 21.1 (typical)	7	3	+	Switzerland	<24 hr old	1 × 105 CFU/larva	21 days	84.85%g
	CH 60 (typical)	7	3	+	Switzerland	<24 hr old	1 × 105 CFU/larva	21 days	91.88%g
[53]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	21 days	approx. 6–20%h
	CH 49.3 (typical)	3	3	Probably +	Switzerland	<24 hr old	3.42 × 104 ± 1.28 × 104 CFU/larva	21 days	approx. 69%h
							4.44 × 104 ± 1.02 × 104 CFU/larva		approx. 90%h
	CH 45.1 (typical)	3	3	−	Switzerland	<24 hr old	1.79 × 104 ± 1.77 × 104 CFU/larva	21 days	approx. 59%h
[76]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	6 days	approx. 3–6%i
	2019BC1 (atypical)	19	12	NI	Canada	<24 hr old	50 CFU/larva	6 days	approx. 26–50%i
							250 CFU/larva		approx. 73–95%i
							500 CFU/larva		approx. 88–99%i
[41]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	6 days	8–18%
	2019BC1 (atypical)	19	12	NI	Canada	<24 hr old	50 CFU/larva	6 days	56–78%
[69]	No infection control	N/A	N/A	N/A	N/A	N/A	0 CFU/larva	6 days	0–16.7%j
	BC (atypical)	19	12	NI	Canada	<24 hr old	~100 CFU/larva	6 days	41.7–66.7%j
	AB (atypical)	19	12	NI	Canada	<24 hr old	~100 CFU/larva	6 days	58.3–91.7%j
	SK (atypical)	36	12	NI	Canada	<24 hr old	~100 CFU/larva	6 days	8.3–100%j

^a Representative findings of previous studies where the genotype of the M. plutonius strain used was specified or known from other papers were summarized. N/A, not applicable. ^b ST, sequence type; CC, clonal complex. Some of the typing results are unpublished observations of Takamatsu et al. ^c +, The strain possessed pMP19 at the time of the experiment; -, The strain did not possess pMP19 at the time of the experiment; NI, no information. ^d CFU, colony forming unit. ^e Since the exact value of the mortality rate was not stated in the reference [55], the approximate value was read from the graphs in Figure 3 in the reference [55]. ^f Since the exact value of the mortality rate was not stated in the reference [40], the approximate value was read from the graphs in Figures 1 and 2 in the reference [40]. ^g Henderson-Tilton corrected mortality calculated by the following formula: 1- ((number of live test bees after treatment * number of live control bees after treatment⁻¹) * (number of live control bees before treatment * number of live bees before treatment⁻¹)) [34]. ^h Since the exact value of the mortality rate was not stated in the reference [53], the approximate value was read from the graphs in Fig 2 (pre-exposure and non-exposed conditions; for the no infection control and CH 49.3) and Fig 4 (non-exposed conditions; for the no infection control and CH 45.1) in the reference [53]. ⁱ Since the exact value of the mortality rate was not stated in the reference [76], the approximate value was read from the graphs in Figure 2 in the reference [76]. ^j Mortalities were calculated using data shown in Table S1 in the reference [69].

Although the virulence of M. plutonius strains/isolates of various origins and genotypes has been examined, the virulence of typical (i.e., CC3 and CC13) and atypical (i.e., CC12) strains has only been compared under the same conditions in studies using Japanese M. plutonius strains/isolates [4, 43, 45]. In these studies, atypical strains always showed higher virulence than typical strains. For example, in a study by Arai et al. [4] in which the bacteria were ingested by first instar larvae at an inoculation dose of approximately 5 × 10⁴ CFU/larva, all atypical (CC12) strains tested killed more than 70–90% larvae within 5 days, while all typical (CC3 and CC13) strains tested only killed less than 20% of larvae at the same dose (Table 2). As explained below (refer to Virulence factors of atypical M. plutonius), the presence of the virulence plasmid pMP19 is essential for typical (particularly CC3) strains to exert their full virulence [43]; therefore, the low virulence of typical strains reported by Arai et al. [4] may have been due to the loss of the plasmid. pMP19-positive CC3 strains killed more than 80% of infected larvae 21 days after an inoculation with a dose of approximately 1 × 10⁴–1 × 10⁵ CFU/larva [43, 45] (Table 2). However, under the same conditions, the representative Japanese atypical strain (DAT561 of ST12, CC12) consistently killed infected larvae more rapidly than pMP19-positive typical strains, suggesting the higher virulence of atypical (CC12) strains than typical (CC3 and CC13) strains [43, 45].

Although some typical strains isolated in European countries (e.g., CH 49.3, CH 21.1, CH 60, and 4–127) also had the ability to kill more than 70% of infected larvae 12–21 days after inoculation with a dose of approximately 1 × 10³–1 × 10⁵ CFU/larva [34, 40, 53] (Table 2), the majority of the typical strains tested in previous studies showed less than 25% mortality 5–6 days after the inoculation [34, 40, 53]. Furthermore, even when the most virulent typical strains (CH 21.1 and CH 60 of ST7, CC3) were infected at a dose of 1 × 10⁵ CFU/larva, larval mortality rates 6 days after the inoculation were approximately 60% [34]. In contrast, atypical strains isolated in North America killed 70–100% of larvae within 6 days in many tests, even at markedly lower inoculation doses (i.e., 50–500 CFU/larva) [41, 69, 76] (Table 2). These findings further suggest that regardless of the country of isolation, atypical (CC12) strains are more virulent than typical (CC3 and CC13) strains at the individual insect level.

On the other hand, observations of the disease at the colony level revealed a different aspect of the effects of this pathogen on honey bees. In a study conducted in England and Wales over the course of two years [13], Budge et al. investigated the relationship between the extent of damage to bee colonies caused by EFB and the genotype of M. plutonius strains that caused these cases. According to the values of disease ranking calculated from the percentage of the diseased brood, CC3 in typical M. plutonius was the most virulent group in the field, followed by CC12 (i.e., atypical M. plutonius, the most virulent type at the individual insect level) and CC13 in the typical type [13].

In AFB, the virulence of causative Paenibacillus larvae strains at the larval level negatively correlated with virulence at the colony level [28, 54]. Exposure bioassays showed that all larvae infected with P. larvae strains of the ERIC II genotype were killed within approximately seven days [28,29,30]; therefore, most of the infected larvae in the field are considered to die before their brood cells are capped, resulting in the efficient removal of the infected larvae from the colony by hygienic nurse bees and a low level of spore production within the colony. In contrast, strains of the ERIC I genotype were less virulent for the individual larva than ERIC II strains in terms of the time course of mortality and required approximately 12 days to kill all infected larvae [28,29,30]; therefore, more larvae died after cell capping in ERIC I strain-infected colonies, leaving nurse bees only a small chance of cleaning out the infected larvae, which resulting in a higher level of spore production, the faster spread of spores within the colony, and faster colony collapse than with ERIC II strain-infected colonies [28, 54].

Similar negative correlations may be present in EFB. In in vitro exposure bioassays conducted by Nakamura et al. [45], all representative CC12 strain-infected larvae died at quite a small size before pupation, whereas some CC3 strain-infected larvae pupated. Therefore, in CC12 strain-infected colonies, most of the infected brood may develop EFB and die at the larval stage before the cell is capped and, thus, are efficiently ejected from the colony by nurse bees, resulting in a rapid reduction in the dose of M. plutonius and a delay in disease dissemination in the colony. In contrast, in CC3 strain-infected cases, more larvae may survive and pupate than CC12 strain-infected cases and, thus, may leave more infective M. plutonius in their fecal deposits in brood cells. In the process of pupation, honey bee larvae secrete a continuous silken fiber from the silk glands to spin a cocoon, and their feces are discreetly stored between the threads of the cocoon. However, infected larvae have poorly developed silk glands, and thus, contaminated feces cannot be enclosed between the layers of the silk cocoon [75], which further facilitates bacterial dissemination and results in more serious disease at the brood frame and colony levels. However, further research is needed to verify this hypothesis.

PATHOMECHANISM OF EFB CAUSED BY ATYPICAL M. PLUTONIUS

As shown above, atypical M. plutonius strains are extremely virulent in honey bee larvae. The infectious cycle of EFB begins when a larva eats brood food contaminated with M. plutonius (Fig. 4A) [59]. According to the study by Takamatsu et al. [66], the ingested cells of atypical strains initially localized to the surface of the peritrophic matrix (PM) in the midgut (Fig. 4B) and begin to proliferate on the PM. As the number of M. plutonius cells increases, the bacterial mass extends towards the lumen (Fig. 4C) and eventually almost completely occupies it (Fig. 4D). The PM is degraded during the course of infection, and M. plutonius directly interacts with the midgut epithelium (Fig. 4E) (also refer to Virulence factors of atypical M. plutonius). Midgut epithelial cells degenerate in infected larvae; however, the infection of atypical M. plutonius is essentially confined to the digestive tract, and the bacteria do not invade or proliferate in the body cavity actively (Fig. 4E) [66]. Most infected larvae eventually die. Nutrient exploitation associated with bacterial growth in the midgut may be one of the factors leading to the larvae’s death; however, the direct cause of death remains unclear. In the field, dead larvae decompose under the effects of secondary invaders, such as Enterococcus faecalis and Paenibacillus alvei, which are present in the bee’s habitat (Fig. 4F).

Fig. 4.

Pathogenesis of European foulbrood caused by atypical Melissococcus plutonius. Microphotographs of infected larvae used in this figure come from those used in the previous study by Takamatsu et al., 2016 [66]. (A) Honey bee larvae are orally infected with M. plutonius by eating food contaminated with the pathogen. (B–D) Ingested atypical M. plutonius cells localize to the surface of the peritrophic matrix (PM) in the midgut and begin to proliferate on the PM. As the number of M. plutonius cells increases, the bacterial mass extends towards the lumen and eventually almost completely occupies it. (E) The PM is degraded during the course of infection due to the PM-degrading proteins produced by the infected atypical strains, and M. plutonius directly interacts with the midgut epithelium. The degeneration of epithelial cells is also observed in the infected larvae, most of which eventually die. (F) Dead larvae decompose under the effects of secondary invaders.

VIRULENCE FACTORS OF ATYPICAL M. PLUTONIUS

The only factor shown to be involved in M. plutonius virulence is pMP19, an approximately 19–20-kbp plasmid that encodes the putative virulence factors, melissotoxin A and a putative extracellular matrix-binding protein [17, 51, 52]. This plasmid is found in some M. plutonius strains regardless of their genotypes [17, 43, 64]. pMP19 is considered to be an essential virulence factor in CC3 strains because the loss of pMP19 abrogated the pathogenicity of strains in exposure bioassays [34, 43] (Table 2). M. plutonius has been described as a pathogen that easily loses its virulence when cultured artificially in laboratory media in vitro [7, 42], and this phenomenon has been attributed to the loss of pMP19 because the plasmid was not stably maintained in M. plutonius during in vitro propagation [43].

However, in contrast to CC3 (typical) strains, strain DAT561 of CC12 (a representative strain of atypical M. plutonius) retained the ability to kill most of the infected A. mellifera larvae in vitro even after the loss of pMP19, indicating that pMP19 is not an essential factor for atypical strains to exert their pathogenicity [43] (Table 2). This finding is consistent with the phenomenon of atypical strains maintaining their virulence even after repeated subcultures in laboratory media [4] and suggests the presence of unidentified pathomechanisms functioning in the virulence of atypical strains [43].

As explained above, the PM in the larval midgut is degraded during the course of atypical M. plutonius infection, and the pathogen directly interacts with the midgut epithelium. In AFB, the causative agent P. larvae also proliferates in the midgut and degrades the PM [28, 79]. The degradation of the PM is a key step in the pathogenesis of AFB, and the PM-degrading protein PlCBP49 has been identified as a key virulence factor of P. larvae [26]. Since the highly virulent atypical strain DAT561 also encodes putative PM-degrading proteins (enhancin, a chitin-binding domain-containing protein, and endo-α-N-acetylgalactosaminidase) [17, 44], PM degradation by these proteins was also considered to be a key step in the pathogenesis of EFB. The low-virulence M. plutonius strain of CC13 (LMG 20360) did not have the ability to damage the PM in bee larvae [5], which further supported this hypothesis. Since the deletion of all genes encoding putative PM-degrading proteins from DAT561 abrogated the ability of the strain to degrade and breach the PM, all or some of the proteins were involved in PM degradation [44]. However, even without the degradation of the PM (i.e., even without a direct interaction between the mutant and midgut epithelium), midgut epithelial cells degenerated over time, and the mutant killed approximately 70–80% of the bee brood. These findings suggest that the PM-degrading proteins are dispensable virulence factors in atypical M. plutonius [44]. No crucial virulence factors for atypical M. plutonius have been identified to date.

Grossar et al. previously reported that M. plutonius virulence correlated with its growth dynamics in artificial medium [34]. Although they did not use atypical strains in their study, this finding may be pertinent. Atypical strains have been shown to form larger colonies on agar media [4] and grow more rapidly in liquid media than typical strains (Takamatsu et al., unpublished observations). Moreover, among three representative strains from CC3, CC12, and CC13, the CC12 (atypical) strain proliferated more vigorously in larvae than the CC3 and CC13 (typical) strains [45]. This vigorous growth ability of atypical strains may be one of the important factors contributing to their high virulence. Even if this hypothesis is correct, there must be gene(s) that endow this vigorous growth ability to atypical strains. A recent genome analysis of M. plutonius strains identified genes unique to a representative atypical strain [17], among which there may be genes involved in its vigorous growth ability and high virulence. However, further studies using gene-manipulated mutants are needed to clarify the true role of each gene in the pathogenesis of EFB.

DETECTION AND DISCRIMINATION OF ATYPICAL M. PLUTONIUS BY PCR

To obtain a detailed understanding of infectious diseases and develop appropriate countermeasures, it is important to identify the type of strain infesting the area and causing each individual case. However, the isolation and characterization of causative agents by standard culture methods are both time-consuming and labor-intensive, and, thus, PCR, a rapid and easy method to detect and identify the causative agents of infectious diseases, has been widely utilized for these purposes in many diseases. Many PCR assays, including regular, hemi-nested, and real-time PCRs, have been developed and utilized for EFB; however, most were able to detect or identify M. plutonius, but not specifically distinguish atypical (CC12) strains from typical (CC3 and CC13) strains [12, 15, 20, 27, 32, 47, 56, 62, 71].

The first PCR assay designed to detect and differentiate typical and atypical M. plutonius strains was the duplex PCR developed by Arai et al. [3]. In this assay, the Na⁺/H⁺ antiporter gene (napA) and Fur family transcriptional regulator gene were selected as targets for typical and atypical M. plutonius, respectively, based on the findings of a comparative genomic analysis result of M. plutonius strains, and two primer sets designed to give type-specific PCR products of easily distinguishable sizes were used [3]. Using the same target genes, Nakamura et al. developed real-time PCR assays that separately quantify typical and atypical strains in infected larvae [45], and Okamoto et al. improved the duplex PCR of Arai et al. [3] and developed a novel multiplex PCR assay that detects and types strains of all foulbrood pathogens (typical and atypical M. plutonius and ERIC I, II, and III-V P. larvae) [49]. These PCR assays have been used not only for the rapid diagnosis of and basic research on EFB caused by atypical M. plutonius, but also to survey the prevalence of atypical strains in several countries as described below [3, 14, 45, 49].

GLOBAL DISTRIBUTION OF ATYPICAL M. PLUTONIUS

Although the global distribution of atypical strains has not yet been examined in detail, atypical M. plutonius has been isolated in geographically distant countries, including Japan, the UK, the Netherlands, Switzerland, the USA, Canada, Mexico, and Brazil, according to the MLST database (https://pubmlst.org/organisms/melissococcus-plutonius [accessed on June 12th, 2023]) and previous studies [3, 4, 13, 14, 33, 35, 36, 41, 64, 65, 67, 69, 76].

Although no large-scale epidemiological studies on EFB have been conducted in Japan, 64 of the 100 M. plutonius strains genotyped to date were assigned to CC12 (i.e., atypical strains) (https://pubmlst.org/organisms/melissococcus-plutonius) [64, 65, 67]. These Japanese atypical strains were mainly isolated from clinical EFB cases, and the atypical type was found not only in European honeybee (A. mellifera), but also in Japanese native honeybee (Apis cerana japonica) [65]. In the study by Arai et al. [3], atypical M. plutonius was detected in 21 of the 35 larval samples collected from clinical EFB cases in Japan. These findings suggest that atypical strains are involved in many EFB cases in Japan. Of the 21 atypical strain-positive larval samples examined in the study by Arai et al. [3], 19 also contained typical strains. The highly frequent coexistence of typical and atypical strains was also observed in Japanese honey. In a study on 116 Japanese honey samples by Okamoto et al. [49], typical and atypical M. plutonius were simultaneously detected in 56.9% of samples. Although 31.9% of honey samples tested were positive for typical M. plutonius alone, no honey had with atypical strains only; therefore, atypical strains always coexisted with typical strains in Japanese honey, suggesting their highly frequent coexistence in apiaries in Japan. The coexistence of the two types was also frequently observed in the State of Chihuahua, Mexico. In the survey conducted by de León-Door et al. [14], M. plutonius was directly detected in 34% (154/448) of the pooled larval samples collected from seven major beekeeping regions of the State by duplex PCR [3]. Among M. plutonius-positive samples, 71% were positive for typical strains only, 3% were positive for atypical strains only, and 26% were positive for both typical and atypical strains [14]; atypical strains were also mostly present with typical strains in Mexico. However, the reasons for the high coexistence of atypical and typical strains in these countries remain unclear.

Although atypical M. plutonius has been isolated in the UK, the Netherlands, and Switzerland, this type appears to be rarer in Europe than in Japan and Mexico. In the survey conducted by Budge et al., atypical (CC12) M. plutonius was involved in only 7 (3.4%) of the 206 EFB cases that occurred in England and Wales between 2011 and 2012 [13]. Similarly, in the survey conducted in Switzerland by Grossar et al., only 4 (2.5%) of the 160 M. plutonius isolates from EFB cases during 2006–2007 and 2013 were assigned to CC12 [35]. All four CC12 strains in Switzerland were isolated from samples in 2013, but not from those during 2006–2007 [35]; therefore, the atypical type may have recently entered Switzerland.

Both types of M. plutonius have been isolated in North America. As of March 2023, six CC3 (typical) strains (5 strains in USA and 1 strain in Canada) and five CC12 (atypical) strains (2 strains in USA and 3 strains in Canada) are included in the MLST database (https://pubmlst.org/organisms/melissococcus-plutonius). However, recent studies conducted in North America reported the frequent isolation of atypical strains. In Canada, Wood et al. isolated the first Canadian atypical M. plutonius from a diseased larva from a blueberry-pollinating colony in 2019 [76]. All M. plutonius isolates isolated from EFB outbreaks in 3 commercial beekeeping operations in western Canada during the summer of 2020 and genotyped by Thebeau et al. [69] were also the atypical type (i.e., ST19 and ST36 strains belonging to CC12). Although not genotyped by MLST, according to a study in 2020 [22], the 16S rRNA gene sequences of all 49 M. plutonius isolates from diseased larvae in the US were the most similar to that of the Japanese atypical strain, DAT561. In addition, one of the US isolates originating from an EFB outbreak in Colorado in 2018 was highly virulent, similar to other atypical strains, and exhibited equivalent growth requirements to those of DAT561 [22], suggesting that all M. plutonius isolates in that study were the atypical type. Furthermore, atypical M. plutonius was isolated from diseased larvae exhibiting the clinical signs of EFB in the survey performed on eight commercial blueberry operations in western Oregon in 2019 and 2020 [33]. As described above, atypical strains were frequently detected in Mexico [14]. In addition, the first possible atypical M. plutonius strains were from samples collected in Brazil [1, 9], and the genotype of a Brazilian strain included in the MLST database <https://pubmlst.org/organisms/melissococcus-plutonius> was ST16 in CC12 (i.e., atypical type). Therefore, the atypical (CC12) type may be the predominant or a major type of M. plutonius in the Americas.

To obtain a more accurate understanding of the global distribution of atypical strains and their impact on the beekeeping industry, epidemiological surveys in different countries using not only samples from clinical EFB cases, but also those from healthy colonies and their environment and a re-examination of M. plutonius strains previously isolated in different parts of the world will be necessary. PCR [3, 49] and real-time PCR [45], which rapidly and easily distinguish atypical strains from typical strains, will be useful in these studies. In previous studies [25, 57], the terms “atypical EFB cases”, “atypical EFB outbreak”, or “atypical form of EFB” have been used. However, these atypical cases do not mean cases caused by atypical strains. “Atypical” in “atypical EFB cases/outbreak” indicates that the clinical signs and course of the cases differ from those of typical (well-known) EFB cases. Therefore, when conducting a literature review on the impact of atypical strains on bee colonies and the distribution of atypical strains in the field, careful checks are needed to establish whether M. plutonius isolates from each case are typed.

REMAINING MYSTERIES AND FUTURE RESEARCH DIRECTIONS

EFB has been attracting attention in recent years, and the number of studies published on EFB and M. plutonius is increasing each year. Although research on atypical M. plutonius has mainly been conducted in Japan [3, 4, 43,44,45, 49, 51, 63,64,65,66,67], atypical strains from North America, particularly those isolated from blueberry-pollinating colonies, have recently been actively investigated [33, 69, 76]. The isolation and distribution of atypical strains in several European countries are also being reported [13, 35, 36]. In this review, I present what has been revealed to date about atypical M. plutonius in light of these studies. However, further studies are warranted on this bacterium. The above section introduced the possibility that atypical strains are not a recently derived new variant, but a long-established type of strain that retains many of the characteristics of its ancestors. Furthermore, atypical M. plutonius has been isolated in geographically distant countries. Therefore, the origin of atypical M. plutonius, the mechanisms by which it has spread worldwide, and how it evaded detection remain unclear. The reasons why the proportion of typical and atypical isolates varies from country to country and why the frequency of atypical strain isolation is increasing in some countries are also unknown. Detailed surveys on the distribution and prevalence of atypical M. plutonius among various honeybee species in various countries, re-examinations of previous isolates and specimens, and genomic analyses of recent and older isolates will provide important insights to help us unravel these mysteries.

As discussed above, damage caused by atypical M. plutonius has been a particular issue in recent years among colonies used to pollinate blueberries in North America [33, 69, 76], and the development of appropriate control methods for atypical M. plutonius is desired. This will require a more detailed understanding of the characteristics of atypical M. plutonius itself and the EFB caused by it; however, the virulence factors of highly virulent atypical M. plutonius and the detailed pathomechanisms of EFB caused by this type are still unknown. With the recent development of genome sequencing technologies, many genes that may be involved in virulence have been identified in M. plutonius in silico [17]. Changes in the bacterial flora with the progression of EFB within insects are also being clarified [2]. However, in silico data alone are not sufficient. Although several genes and a plasmid predicted to be involved in the high virulence of atypical M. plutonius have been removed, none significantly attenuated their virulence [43, 44]. Therefore, in addition to in silico studies, it is necessary to simultaneously conduct experiments using live bacteria and honeybees, such as the construction of gene-manipulated M. plutonius, in vitro and in vivo bioassays of honeybees, and histopathological analyses of infected larvae. In recent years, researchers from various specialties, such as entomology, veterinary medicine, microbiology, gene manipulation, and bioinformatics, have begun to study EFB. By accumulating their techniques and wisdom, more detailed insights into this old and new honeybee disease will gradually be obtained.

CONFLICTS OF INTEREST

The author declares no conflicts of interest associated with this manuscript.