PLoS One. 2015 Feb 6;10(2):e0117200.

Identification of candidate agents active against N. ceranae infection in honey bees: Establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

Sebastian Gisder and Elke Genersch

Institute for Bee Research, Department of Molecular Microbiology and Bee Diseases, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany

 

ABSTRACT

Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

PMID: 25658121

 

Supplementary

Honey bees play an important role as pollinators of crops and fruit in agriculture [1, 2]. Hence, the ongoing decline of managed honey bee (Apis mellifera) populations in several parts of the world and the increasing gap between pollination demand and pollinator availability [3] may have severe implications for food security. In the recent past intense research work has been done in order to identify possible reasons for the decline and consensus now is that it is a multifactorial process with pests and pathogens definitely playing no minor role [4-6]. One emerging pathogen which is reported to cause death of entire colonies is the microsporidium Nosema ceranae [7].

Microsporidia are obligate intracellular parasites that infect a wide range of vertebrate and invertebrate hosts. They are highly evolved fungal parasites [8] which are perfectly adapted to their obligate intracellular lifestyle. Outside the host they exist as metabolic inactive spores (Fig. 1A). Microsporidia infect their host cells by means of the polar tube (Fig. 1A). Subsequent to polar tube extrusion the sporoplasm, which is the actual propagating parasite, is transferred from the spore into the host cell cytoplasm where massive parasite reproduction occurs. Finally, the host cell is filled up with new environmental spores (Fig. 1B) which are released upon lysis of the cells.

 

SG fig1

Figure 1: (A) Nosema ceranae spores using a Raster Electron Microscope (REM) at 5000x magnification. In vitro-germination of spores can be triggered with drying and subsequent rehydration. The polar tube (Pt) is extruded and penetrates the host cell which serves as transfer tube for the sporoplasm. (B) Giemsa stained section of a Nosema ceranae-infected honey bee midgut. Epithelial cells are heavily infected with spores (Sp). 400x magnification, bar represents 30 µm.

 

 

The Western honey bee can be infected by the two microsporidia, Nosema apis and Nosema ceranae. Infections with N. apis were first described in 1909 [9] and frequently reported since then. The clinical symptoms include crawling bees, dysentery, and a shortened life span of the individual adult bee which, however, rarely leads to the collapse of the entire colony. N. ceranae is an emerging pathogen who jumped from its original host, the Eastern honey bee (Apis cerana), to Apis mellifera some decades ago [10-12]. In contrast to N. apis infections, N. ceranae infections in Western honey bees were reported to have a severe impact at both individual and colony level and to result in large scale colony losses. This high virulence of N. ceranae was observed in experiments with caged bees and in the field for colonies mainly in Southern Europe [7, 13]. However, especially the effect of N. ceranae in naturally infected colonies could not be substantiated from other regions of the world [7, 13-19]. In the meantime, N. ceranae jumped host again and now also infects bumblebees with deadly consequences [20, 21]. Taken together, these reports show that there is an urgent need to develop novel treatment strategies to combat N. ceranae in honey bee colonies in order to improve bee health and survival thereby hopefully taking the infection pressure from bumblebees.

The only antibiotic known to be effective against Nosema spp. is fumagillin which has been widely used in the past against N. apis infections [22-25]. However, the use of antibiotics including fumagillin in honey bee colonies is prohibited in Europe. Moreover, the efficacy of fumagillin in sustainable treatment of N. ceranae infections is highly questionable [26]. Hence, there is an urgent need for novel anti-nosemosis substances. So far, the search for such substances had to be performed via infection assays using caged adult bees. However, these experiments are labor intensive, cannot be standardized, and can only be performed during the honey bee season when enough adult bees are available for experiments. State-of-the-art screening assays for identifying substances active against Nosema spp. based on cultured cells were lacking thus hampering the progress in the treatment of Nosema spp. infections of honey bee colonies. This situation changed when we established an in vitro-infection model for honey bee pathogenic Nosema spp. using the heterologous lepidopteran cell line IPL-LD 65Y derived from the gypsy moth Lymantria dispar [27]. For further developing this cell culture model into an in vitro screening assay, a protocol for quantifying Nosema spp. propagation in infected cell cultures was needed as well as a format allowing automated processing of the assays. To this end, we first analyzed the temporal gene expression pattern of seven annotated microsporidial genes in infected cells using RT-PCR. The results showed that the gene expression of the annotated polar tube protein 2 (acc. no. EQB61988.1) of N. apis was most suitable to be used for molecular detection of an ongoing in vitro-infection for both, N. apis and N. ceranae. The gene expression of ptp2 was first detectable 20 hours post infection and was continuously detected until the end of the observation period at 72 hours post infection. For quantitative analysis of ptp2 gene expression we adapted the recently published protocol for Leishmania parasite quantification [28] and established a medium throughput assay which can be performed in 96-well microtitre plates thus allowing automation of the assay in the future. Briefly, the cells were in vitro-infected with N. ceranae and were subsequently transferred into the microtitre plate together with the test substances with known or assumed activity against N. ceranae. After 72 hours all cells were removed from the wells and total RNA was extracted. Next, gene expression of the ptp2 gene was verified with RT-PCR using a primer set with one primer labelled with digoxigenin and the other primer labelled with biotin (Fig. 2).

 

 SG fig2

Figure 2: Principle of the RT-PCR-ELISA technique used in this study (Figure modified from (28)). RNA was extracted from (treated) infected cells. After RT-PCR the putative PCR product presents digoxigenin (green) and biotin (yellow). The PCR product was transferred into the microtitre-plate and biotin binds to streptavidin which is coated to the well. After washing the well, all unbound components were removed and an anti-digoxigenin antibody which is coupled to a peroxidase binds to the presented digoxigenin. The substrate ABTS is converted by the peroxidase into a visible blue color.

 

 

The PCR products were then transferred to the streptavidin coated wells of a microtitre plate. PCR products with incorporated, biotin-labelled primers were linked to the plate via streptavidin-biotin interactions. Linked PCR-products also carrying a digoxigenin-labelled primer were detected via an peroxidase-coupled, anti-digoxigenin antibody. The subsequent blue color reaction in the wells of the microtitre plate could be quantified by using an ELISA reader (Fig. 3A). This protocol ensured that only PCR-products carrying both labels and, hence, being indicative for ongoing pathogen proliferation, were detected via this PCR-ELISA.

We first confirmed the robustness and reliability of our system with fumagillin, the antibiotic known for its activity against Nosema spp. infections in honey bees. Expression of ptp2 could be detected in infected cells as indicated by a strong blue color reaction, while no ptp2 expression was evident in infected and fumagillin treated cells as revealed by the lack of any color reaction in the respective wells of the microplate (Fig. 3). Quantification of the color reaction by an ELISA reader is advisable and was performed resulting in arbitrary units for steady state levels of ptp2 mRNA. While no gene expression for ptp2 could be measured in mock infected cells, N. ceranae infected cells showed high levels of ptp2 gene expression (Figure 4A). In infected cells that were treated with fumagillin (0.01 mg/ml), proliferation of N. ceranae was inhibited so that only residual gene expression of ptp2 was detectable (Fig. 4A). Microscopic analysis (Fig. 4B) confirmed these results and revealed no vegetative microsporidian forms in the cytoplasm of the fumagillin treated infected cells while in infected, untreated cells early spores were observed. Hence, the newly developed in vitro-test system reliably differentiated between infected non-treated cells and infected fumagillin-treated cells and substantiated the activity of fumagillin against N. ceranae also in cell culture.

 

SG fig3

Figure 3: (A) Detection of infected IPL-LD 65Y cells with the RT-PCR-ELISA protocol. In mock infected cells no N. ceranae ptp2 gene was amplified during PCR-reaction. No color reaction was observed. In N. ceranae infected cells a strong color reaction was detected with the ELISA Reader. Only residual color reaction was detectable in the wells with fumagillin (0.01mg/ml) treated infected cells. (B) The microtitre plate was analyzed with an ELISA-reader using 405 nm excitation wavelength.

 

SG fig4

Figure 4: (A) Results of the evaluation of the RT-PCR-ELISA medium throughput system. Whereas no gene expression of N. ceranae polar tube protein 2 (ptp2) was detectable in mock infected cells, there was a clear signal for ptp2 in infected cells 72 hours post infection. In infected cells which where treated with fumagillin (0.01 mg/ml) no signal for a successful infection could be detected. All columns represent mean values ± SD of three independent replicates per group. Columns with different letters differ significantly, while columns with the same letter are not significantly different (ANOVA with Kruskal-Wallis nonparametric multiple comparison test with α<0.05). (B) Microscopic confimation of the molecular assay shows that no spore or vegetative form of N. ceranae was detectable in infected cells after treatment with fumagillin (0.01 mg/ml). Bars represent 10 µm.

 

Next, we tested the effect of several putatively inhibitory substances like paramomycin, albendazole, ornidazole, tinidazole, metronidazole, quinine, and surfactin. Additionally to the ptp2 gene expression, we determined the cytotoxic effect of every substance in any used concentration for ruling out that negative results were due to cytotoxic effects rather than to inhibition of infection. We further confirmed all RT-PCR-ELISA obtained results by microscopic analyses of Giemsa-stained cells. Negative RT-PCR-ELISA results always correlated with negative microscopic results, hence with no vegetative forms of N. ceranae detectable in cultured cells. Amongst the analyzed substances, only the nitroimidazoles tinidazole (2 mg/ml) and metronidazole (2 mg/ml) showed a significant reduction in the steady state level of the ptp2 mRNA levels (Fig. 5), no vegetative forms of N. ceranae inside Giemsa-stained cells and only weak concurrent cytotoxicity.

 

SG fig5

Figure 5: Effect of putative anti-Nosemosis substances against N. ceranae in in vitro-infected IPL-LD-65Y cells. DMSO served as solvent for all substances and showed no activity against in vitro N. ceranae-infection. All substances showed only weak cytotoxicity when presented concentrations were used. Expression levels of the N. ceranae ptp2 gene in infected IPL-LD 65Y cells were determined via the presented RT-PCR-ELISA protocol. Cells were infected with N. ceranae and then directly treated with DSMO, paromomycin, albendazole, ornidazole, tinidazole, metronidazole, quinine, and surfactin in the given concentrations. Results of the RT-PCR-ELISA for steady state levels of ptp2 mRNA were expressed in arbitrary units. All columns represent mean values ± SD of three independent replicates per group. Statistical analysis was performed with ANOVA and Kruskal-Wallis nonparametric multiple comparison test with α<0.05. Columns with different letters differ significantly, while columns with the same letter are not significantly different.

 

This indicates that metronidazole and tinidazole are able to inhibit N. ceranae infections as effective as fumagillin and could, therefore, serve as alternative anti-nosemosis drugs. However, both nitroimidazoles are antibiotics and regarding to the European Union (Commission Regulation (EU) No 37/2010 of 22 December 2009) antibiotics particularly nitroimidazoles are prohibited in animal food and food animals [29]. That is why both substances will not have any future in the treatment of Nosema spp.-infected honey bees but may be in the treatment of infected bumblebees produced for and used in pollination in greenhouses. In conclusion, our novel cell culture based and RT-PCR-ELISA coupled medium-throughput screening assay for putative anti-nosemosis substances is now available for screening further candidate substances or entire substance libraries. This will finally result in the identification of novel drugs improving honey bee and bumblebee health.

Importance of this study: Our medium throughput screening assay now provides a so far unavailable means to test putative anti-Nosemosis substances in vitro. This powerful tool makes the search for active substances independent from extensive in vivo infection experiments and the honey bee season and potentiates the number of substances that can be tested in a reasonable time frame.

 

 

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Acknowledgements

This work was supported by grants from the Ministries for Agriculture from the Federal States of Brandenburg and Sachsen-Anhalt, Germany, and by a grant (511-06.01-28-1-34.007-07) from the German Ministry of Nutrition, Agriculture and Consumer Protection (BMELV). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

 

Contact

PD Dr. Elke Genersch

Institute for Bee Research

Friedrich-Engels-Str. 32

Germany 16540 Hohen Neuendorf

Phone: 03303/2938-30

Fax:     03303/2938-40

elke.genersch@hu-berlin.de

 

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