European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent,
Melissococcus plutonius
, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although
M. plutonius
is believed to be remarkably homologous, in addition to
M. plutonius
isolates with typical cultural characteristics,
M. plutonius
-like organisms, with characteristics seemingly different from those of typical
M. plutonius
, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14
M. plutonius
and 19
M. plutonius
-like strain/isolates revealed that, unlike typical
M. plutonius
strain/isolates,
M. plutonius
-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only
M. plutonius
-like isolates, but not typical
M. plutonius
strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that
M. plutonius
-like organisms were taxonomically identical to
M. plutonius
. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (
M. plutonius
-like) isolates were separately grouped into two genetically distinct clusters. Although
M. plutonius
is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical
M. plutonius
maintained the ability to cause EFB in honeybee larvae even after cultured
in vitro
in laboratory media. Because the rapid decrease of virulence in cultured
M. plutonius
was a major impediment to elucidation of the pathogenesis of EFB, atypical
M. plutonius
discovered in this study will be a breakthrough in EFB research.
European foulbrood is a contagious bacterial disease of honey bee larvae. Studies have shown that the intestinal bacteria of insects, including honey bees, act as probiotic organisms. Microbial flora from the gut of the Japanese honey bee,
Apis cerana japonica
F. (Hymenoptera: Apidae)
,
were characterized and evaluated for their potential to inhibit the growth of
Melissococcus plutonius
corrig. (ex White) Bailey and Collins (Lactobacillales: Enterococcaceae)
,
the causative agent of European foulbrood. Analysis of
16S rRNA
gene sequences from 17 bacterial strains isolated by using a culture-dependent method revealed that most isolates belonged to
Bacillus, Staphylococcus,
and
Pantoea.
The isolates were screened against the pathogenic bacterium
M. plutonius
by using an in vitro growth inhibition assay, and one isolate (Acja3) belonging to the genus
Bacillus
exhibited inhibitory activity against
M. plutonius.
In addition, in vivo feeding assays revealed that isolate Acja3 decreased the mortality of honey bee larvae infected with
M plutonius,
suggesting that this bacterial strain could potentially be used as a probiotic agent against European foulbrood.
Melissococcus
plutonius is the causative agent of an important honeybee disease, European
foulbrood (EFB). In addition to M. plutonius strains with typical
characteristics (typical M. plutonius), we recently reported the presence
of atypical M. plutonius, which are phenotypically and genetically
distinguished from typical M. plutonius. Because typical and atypical
M. plutonius may have different pathogenic mechanisms, differentiation
of these two types is very important for diagnosis and more effective control of EFB. In
this study, therefore, a duplex PCR assay was developed to detect and differentiate
typical and atypical M. plutonius rapidly and easily. On the basis of the
results of comparative genomic analyses, we selected Na+/H+
antiporter gene and Fur family transcriptional regulator gene as targets for detection of
typical and atypical strains, respectively, by PCR. Under optimized conditions, the duplex
PCR system using the designed primers successfully detected and differentiated all typical
and atypical M. plutonius strain/isolates tested, while no product was
generated from any other bacterial strains/isolates used in this study, including those
isolated from healthy honeybee larval guts. Detection limits of the PCR were 50 copies of
chromosome/reaction for both types, and it could detect typical and atypical M.
plutonius directly from diseased honeybee larvae. Moreover, the duplex PCR
diagnosed mixed infections with both M. plutonius types more precisely
than standard culture methods. These results indicate that the duplex PCR assay developed
in this study is extremely useful for precise diagnosis and epidemiological study of
EFB.
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