• 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.

• International Journal of Industrial Entomology and Biomaterials
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• v.12 no.2
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• pp.87-93
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• 2006

Multiplication and spore production of three microsporidia viz., Nosema bombycis, Nosema sp. 1 and Nosema sp. 2 in fifth instar larval tissues of silkworm, Bombyx mori L. in two seasons with distinct temperature regimes were studied. Nosema sp. 2 produced significantly (P < 0.01) higher number of spores in various tissues. Among the tissues, spore production was highest in silk gland, followed by fat body and gut. Spore production was significantly (P < 0.01) higher in season-II (Average temperature $29.4{\pm}1.1^{\circ}C$). Maximum spore production was observed 25 days post inoculation (p. i.) in season-I (Average temperature $18.9{\pm}1.1^{\circ}C$), whereas in season-II, it was 14 days p. i. In season-I, spore production was low up to 21 days p. i., then increased sharply. In season-II, there was a steady increase in spore production. The results indicate that the microsporidian multiplication is tissue specific and extremely sensitive to temperature at which the host is reared. It also reveals that, silk gland, fat body and gut are the most appropriate tissues for microscopic identification of microsporidia in the larval stage.

• International Journal of Industrial Entomology and Biomaterials
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• v.19 no.2
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• pp.221-228
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• 2009

Nosema sp. infection in the Indian tasar silkworm, Antheraea mylitta exerts a complex of influences on its host. The instar duration was extended significantly (P<0.001) except in $1^{st}$ instar. The infected larvae took about 48 days to reach the spinning stage against 40 days in the uninfected ones. The final weight attained by the larva at the end of each instar of development declined significantly following infection, as did weight gain and relative growth rate (RGR). The growth recorded/ day declined in infected larvae compared to uninfected ones from 8.2% during $1^{st}$ instar to 43.3% during $5^{th}$ instar. Food ingestion and digestion increased with advancement of the instar significantly irrespective of the status of the larvae but the relative consumption rate (RCR) declined. These parameters significantly declined in infected larvae (except food digested during $2^{nd}$ instar). The decline was more during $3^{rd}$ instar. In contrast, the approximate digestibility (AD %) was significantly higher in infected larvae than uninfected ones leaving the $1^{st}$ instar larvae unaffected. The efficiency of conversion of ingested food (ECI) and efficiency of conversion of digested food (ECD) did not change in a patterned way following the microsporidia (Nosema sp.) infection. The values of ECI significantly changed during $2^{nd}$, $3^{rd}$ and $5^{th}$ instars; while the change in ECD during $2^{nd}$, $4^{th}$ and $5^{th}$ were significant. During the entire larval life all the parameters tends to decline significantly following microsporidia infection but AD registered a significant increase. Nosema sp. spore concentration has increased 270.7 times during larval development in the course of experimentation.

• Journal of Sericultural and Entomological Science
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• v.38 no.1
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• pp.48-52
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• 1996

Nosema bombycis was found on the both of surface and inside of silkworm eggs layed by infected female moth, maternal-mediated transmission of those pathogens via the surface or internal site of silkworm eggs were investigated. All of the meconia from infected female moths contained pathogenic spores, those concentration were 4.6(${\pm}$0.24)X106 /ml. The pathogens on the surface of nondiapause eggs were transmitted to the progeny larvae at the rate of 54.5%, however, lost their activity before hatching for the case of overwintered eggs or acid treatment(16% HCI at 46.2$^{\circ}C$ for 6 min). N. bombycis in the silkworm eggs localized at the serosa, yolk, embryo except chorion tissue in the silkworm eggs layed by infected female moths. Development of the pathognes in the eggs was synchronized with embryogenesis, which secured the safety of pathogens against environmental condition, resulting in the high transmissibility of 91.2${\pm}$1.80%.

• Korean Journal of Veterinary Research
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• v.16 no.2
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• pp.165-171
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• 1976

Experimental approaches on the effectiveness of thimerosal to control growth of Nosema apis (Zander, 1909) were carried out in the rearing honey bees. The rearing honey bees were artificially infected with various levels of spore isolated from local honey bees. The results obtained were summarized as follows: 1. In the experiments of therapeutic chemicals for Nosema disease, 0.01% and 0.02% thimerosal of sucrose-honey mixture was the most effective agent but the each concentration of amprolium, furazolidone, hygiene, sulfadimethoxine and terramycin did not show the any effects 2. It showed very high therapeutic effectiveness (over than 90%) that the treatment of three times every other day after the treatment of three times consecutive every day with 0.01% thimerosal, or the treatment of three times consecutive every day with 0.02% thimerosal. 3. When 0.02% thimerosal was administered three times consecutive every day to honey bees at the 4th day before artificial inoculation of N. apis, it showed very high degree (100%) of prevalence control effectiveness, and it also showed high degree (over than 90%) in administration at the 7th day before, and over than 80% at the 10th day before. Then authors found that thimerosal has the prevalence control effectiveness as well as the treatment effectiveness. 4. In the rearing honey bee colony, 0.02% thimerosal showed the high degree (over than 80%) of therapeutic effectiveness with the various levels which contained from the light decree of infection to the severe degree of it.

• International Journal of Industrial Entomology and Biomaterials
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• v.26 no.1
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• pp.1-12
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• 2013

The commercial rearing of polyphagous Indian tasar silkworm, Antheraea mylitta Drury being practiced on naturally grown primary food plants like Terminalia arjuna, (Arjun) Terminalia tomentosa (Asan), and Shorea robusta (Sal) available in the tropical forests of central India, at times, is seriously affected by the disease- Pebrine, caused by Nosema sp., a microsporidian pathogen. The present investigation on comparative larval, silk gland weight and also cocoon parameters in Pebrine-free and Pebrine-infected ecorace of tasar silkworm Antheraea mylitta Drury (Daba TV), illustrates the tasar silkworm larvae infected with pebrine disease causing heavy losses to the economy of the silk industry.

• Korean Journal of Veterinary Research
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• v.16 no.1
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• pp.11-25
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• 1976

Studies on pathogenicities and developmental stages of Nosema apis (Zander, 1909) were carried out through artificial infection to Nosema free honey bees with various levels of spores isolated from local honey bee colony. The results obtained were summarized as follows: 1. The clinical symptoms were observed as dysentery, enteritis of mid-gut (enlargement and decoloration), crawling posture and shortening of the longevity of worker bees in the rearing honey bee colony inoculated with the spores. 2. Number of spores harvested from laboratory rearing honey bees were progresively increased to 4 weeks after inoculation. The regression equations and coefficients of correlations to various spore levels were as follows in each treatment colony. Colony 1. ($$1,000{\times}10^4spores/ml$$) $$y_{c1}=471{\times}10^{4}x+454{\times}10^4(r=0.65^*$$) Colony 2. ($$500{\times}10^4spores/ml$$) $$y_{c2}=340{\times}10^{4}x+207.8{\times}10^4(r=0.99^{**}$$) Colony 3. ($$100{\times}10^4spores/ml$$) $$y_{c3}=150{\times}10^{4}x+84.2{\times}10^4(r=0.99^{**}$$) Colony 4. ($$10{\times}10^4spores/ml$$) $$y_{c4}=13.8{\times}10^{4}x+13{\times}10^4(r=0.98^{**}$$) 3. Average longevity of worker bees artificially infected with Nosema apis was shortened as 21.7~43.8% compare to the control. (p<.05, p<.01) 4. The spores which were isolated from honey bee colony infected with Nosema disease were ovoid or spherical form, and measured, as a rule, from $4.7{\mu}m$ to $6.1{\mu}m$ (mean $5.3{\mu}m$) in length and from $2.4{\mu}m$ to $3.2{\mu}m$ (mean $2.9{\mu}m$) in width. 5. In the mid-gut of honey bees, the spore was progresively germinated and became trophozoite stage. The trophozoites were grown to meronts and their binary fission were begun. The divided two sporoblasts were developed to the spores which had elastic membrane. The new spores were shed in excreta of honey bees 10~15 day after inoculation at $25{\pm}2$ centigrade. 6. The ultrastructure of spore membrane consisted of three layers, such as, outer, middle and inner layer. The sporoplasm consisting lamellar structure occupied only anterior part of the spore and was often extended to posterior direction where definite vacuoles and a polar filament was able to detect.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.04a
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• pp.69-69
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• 2003

We took the nationwide survey of three main honeybee diseases, European foulbrood (EFB; Melissococcus pluton), chalk brood (CB; Ascosphera apis), and nosema (Nosema spp.) in 2001 and 2002 from South Korea. The number of infected apiaries with EFB and CB examined from 21 apiaries were 9 and 13, respectively. The average percentages of infectedcolonies in apiaries where EFB and CB occurred were 7.4% and 12.8%. (omitted)

• Journal of Sericultural and Entomological Science
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• v.36 no.1
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• pp.69-75
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• 1994

Through the histological and anatomical investigation of silkworm gonad, N. bombycis infection was found to begin from the peripheral region of ovarial sheath or testicular sheath, then, the pathogens spread to the inner portion. Peroral inoculation with purified spores of N. bombycis to 2nd instar larvae at dosages around 106-8/㎤ of artificial diet resulted in the extremely extended larval survival as long as 15 to 22 days of 4th instar. The growth of ovarioles was confirmed in the 10 to 14 day old larvae, oogonia developed into oocytes and nurse cell against heavy infection of the ovary. Gonads rarely obstructed oogenesis and spermatogenesis in the pupae failed in adult eclosion. Light infection of female hosts effected insignificantly on the ovarial development, however, recorded 100% transmission of the pathogens to the progeny populations. Conclusively, ovarial inflection of silkworm induce transovarial transmission begins around 2~3 day old pupae when ovarioles extruded out to hemocoel, and the infection period thought to be continued until the stage of eggs complete shell formation in the ovariole.

• 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.