• Journal of Apiculture
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• v.34 no.2
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• pp.137-140
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• 2019

Although honeybees (Apis mellifera) are crucial for maintenance of the ecosystem, population of honeybee has been steadily decreasing due to diseases including nosemosis. Nosemosis is a disease caused by Nosema ceranae and is now considered as a major threat to honeybees. N. ceranae is a microsporidian that stays in form of spore even before the infection, which makes it harder to control than other pathogens. People are now aware of this parasite, however, cure and preventive candidates for nosemosis are hardly found until today. In this study, in vitro experiment of Lespedeza cuneata treatment to prevent nosemosis were done using Trichoplusia ni cell line, BTI-TN5B1-4. Normal T. ni cells exhibited round shape without abnormal size. On the other hand, when N. ceranae were treated, cells deteriorated and some cells abnormally enlarged due to N. ceranae infection. Interestingly, treatment of T. ni cells with L. cuneate extract protected abnormal cell shape induced by N. ceranae infection to normal shape. Some N. ceranae spores were observed outside of the cells. Effective concentration range for N. ceranae control were experimented. Lowest concentration which can control nosemosis were 50 ㎍/mL. When the concentration of L. cuneata extract was exceeded 200 ㎍/mL, cytotoxicity started to show up.

• Korean Journal of Microbiology
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• v.49 no.3
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• pp.270-274
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• 2013

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators since the outbreak of honeybee collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites, which affect the life span and fecundity of their host, have been discovered in B. terristris. In order to detect the microsporidian pathogen, Nosema spp. in the field populations of B. terristris, we collected adults and isolated their genomic DNA for diagnostic PCR. The PCR primers specific for Nosema spp. were newly designed and applied to gene amplification for cloning. Only small subunit ribosomal RNA (SSU rRNA) gene of N. ceranae was successfully amplified among examined genes and sequenced, which indicates that N. ceranae mainly infects the examined field population of B. terristris. To detect of SSU rRNA gene, two regions of SSU rRNA gene were selected by primary PCR analysis and further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that SSU rRNA of N. ceranae was detected at concentration as low as $0.85ng/{\mu}l$ genomic DNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae infection in the field population as well as risk assessment of B. terristris.

• Mycobiology
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• v.45 no.3
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• pp.204-208
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• 2017

Nosema ceranae is an obligate intracellular fungal parasite that causes mortality in honey bees and enhances the susceptibility of honey bees to other pathogens. Efficient purification of Nosema spores from the midgut of infected honey bees is very important because Nosema is non-culturable and only seasonably available. To achieve a higher yield of spores from honey bees, in this study, we considered that the initial release of spores from the midgut tissues was the most critical step. The use of 2 mm beads along with enzymatic treatment with collagenase and trypsin enhanced the homogenization of tissues and the yield of released spores by approximately 2.95 times compared with the use of common 3 mm beads alone. The optimal time for the enzyme treatment was determined to be 1 hr as measured by the yield and viability of the spores. A one-step filtration using a filter paper with an $8-11{\mu}m$ pore size was sufficient for removing cell debris. This method may be useful to purify not only N. ceranae spores but also other Nosema spp. spores.

• Journal of Veterinary Science
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• v.22 no.3
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• pp.40.1-40.12
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• 2021

Background: The microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen. Objectives: The present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees. Methods: A procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration. Results: UR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min. Conclusions: UR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2023.04a
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• pp.6-6
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• 2023

Nosemosis is one of the most common protozoan diseases of adult bees (Apis mellifera). Nosemosis is caused by two species of microsporidia; Nosema apis and Nosema ceranae. Nosema ceranae is potentially more dangerous because it has the ability to infect multiple cell types, and it is now the predominant microsporidian species in A. mellifera. In this study, we identified two anti-nosemosis plants, Aster scaber and Artemisia dubia, which reduced the spore development of N. ceranae in spore-infected cells. We intend to establish the anti-nosemosis activity of aqueous, ethyl acetate (EA), and butanol (BuOH) extracts of A. dubia and A. scaber. In order to determine the optimal dose, we did in vitro and in vivo toxicity for all the extracts and carried out anti-nosemosis experiments. Although all of the extracts (aqueous, EA, and BuOH) showed in vitro and in vivo anti-nosemosis activity in a dose-dependent manner, the aqueous extracts of A. dubia and A. scaber showed more potent anti-nosemosis activity than the EA and BuOH extracts. And then, we isolated five phenolic compounds [chlorogenic acid, 3,4-dicaffaeoylquinic acid (3,4-DCQA), 3,5-dicaffaeoylquinic acid (3,5-DCQA), 4,5-dicaffaeoylquinic acid (4,5-DCQA), and coumarin] from A. dubia, A. scaber, and A. dubia + A. scaber aqueous extracts and screened for their toxicities and anti-Nosema effects in both in vivo and in vitro conditions. Among these five compounds, coumarin, chlorogenic acid, and 4,5-DCQA exhibited less toxic but more potent anti-Nosema effects than the other two compounds. Especially, chlorogenic acid and coumarin showed prominent anti-Nosema activities even at the lowest concentration (10 ㎍/mL). They might have potential to be developed as alternative compounds for the control of Nosema disease.

• Journal of Apiculture
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• v.32 no.1
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• pp.27-39
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• 2017

PCR-chip-based ultra-rapid multiplex PCRs for detection of six major infectious pathogens in honeybee were developed. The 6 kinds of major infectious pathogens in honeybee included Paenibacillus larvae causing American Foulbrood, Melissococcus plutonius causing European Foulbrood as bacteria, Ascosphaera apis (Chalkbrood), Aspergillus flavus (Stonebrood), Nosema apis and Nosema ceranae (Nosemosis) as fungi. The developed PCR-chip-based ultra-rapid multiplex PCR showed successful amplification for all six major pathogens in the presence of more than $10^3$ molecules. The time for confirming amplification (Threshold cycles; Ct-time) was about 7 minutes for two species, and about 9 minutes for four species. Total 40 cycles of PCR took 11 minutes 42 seconds and time for melting point analysis was 1 minute 15 seconds. Total time for whole PCR detection was estimated 12 minutes 57 seconds (40 cycles of PCR and melting point analysis). PCR-chip based ultra-rapid multiplex PCR using standard DNA substrates showed close to 100% accuracy and no false-amplification was found with honeybee genomic DNA. Ultra-rapid multiplex PCR is expected to be a fast and efficient pathogen detection method not only in the laboratory but also in the apiary field.