Five-Year Cohort Study of Nosema spp. in Germany: Does Climate Shape Virulence and Assertiveness of Nosema ceranae?

Bee samples and field survey.A cohort of 220 colonies kept in 22 apiaries (10 randomly selected colonies per apiary) and managed by hobbyist beekeepers in the northeastern part of Germany (see Fig. S1 in the supplemental material) were monitored for Nosema infection between spring 2005 and spring 2009. The colonies selected for the survey were closely monitored by a professional bee inspector twice a year for the duration of the study without introducing any changes in the beekeeping practices of the beekeepers. Each March, about 100 dead bees were collected from the bottom board of each colony (representing the bees that had died over the winter), and live bees were collected from the winter cluster (representing the bees that had survived the winter). Qualitative Nosema detection and differentiation did not differ between these two samples for a given colony; therefore, the bees collected from the bottom boards were used in the course of the study. At the end of September/beginning of October, around 100 live adult bees were collected from each colony (representing the bees that were raised for overwintering). The collected bees were frozen at −20°C and stored until analysis. Overwintering success, survival during the summer season, and the presence of symptoms of nosemosis were recorded individually for each colony. None of the monitored colonies showed clinical symptoms of nosemosis during the study. Lost colonies were replaced by colonies from the same apiary, preferentially by nuclei made from those colonies in the previous year. Since 220 colonies were observed throughout the study, the sampling of the monitored colonies and of nuclei replacing lost colonies at nine different time points between 2005 and 2009 resulted in a total of 1,997 samples analyzed (see Tables 1 and 2).

Detection of Nosema spp.Qualitative microscopic diagnosis of Nosema spores was performed according to the method described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, published by the Office International des Epizooties (OIE), the World Organization for Animal Health (38a). Briefly, 20 abdomens from each sample were homogenized together in 2 ml double-distilled H₂O (ddH₂O) and were checked by light microscopy (magnification, ×400) for the presence of microsporidian spores (41). The rather moderate sampling size does not allow the detection of the odd infected bee in the colony but does allow the detection of an infection level above ∼15% at the 5% significance level (22), which can be considered biologically relevant (28).

Microscopically positive homogenates were transferred to a 1.5-ml reaction tube and were thoroughly homogenized with a 3-mm-diameter tungsten carbide bead (Qiagen) in a mixer mill (Retsch) for 30 s at 30 Hz. Subsequently, the homogenate was centrifuged at 16,000 × g for 3 min, and DNA was extracted from the pellet using standard methods according to the manufacturer's protocols (Plant DNA extraction kit; Qiagen). The extracted DNA was resuspended in 50 μl elution buffer (Qiagen) and was frozen at −20°C until differentiation.

From each Nosema-positive bee sample, individual bees were dissected under a dissecting microscope (magnification, ×10) under sterile conditions. The tissues (gut, malpighian tubules, fat body, hypopharyngeal glands, and brain) were carefully isolated from individual bees by using fresh forceps for each organ to prevent contamination. The isolated organs were washed twice in 1× phosphate-buffered saline (PBS) and nuclease-free water to wash off any potentially contaminating hemolymph. Subsequently, DNA was extracted from the tissues as described above. The extracted DNA was resuspended in 50 μl elution buffer (Qiagen) and was frozen at −20°C until differentiation.

Differentiation of Nosema spp. via PCR-restriction fragment length polymorphism (PCR-RFLP).A region of the 16S rRNA gene that is conserved for N. apis and N. ceranae (33) was selected for primer design using MacVector, version 6.5 (Oxford Molecular). Primers nos-16S-fw (5′-CGTAGACGCTATTCCCTAAGATT-3′; positions 422 to 444 in GenBank accession no. U97150 [25a]) and nos-16S-rv (5′-CTCCCAACTATACAGTACACCTCATA-3′; positions 884 to 909 in GenBank accession no. U97150 [25a]) were used to amplify ca. 486 bp of the partial 16S rRNA gene.

PCR analysis was performed in a final volume of 25 μl containing 5 μl of template DNA (extracted from pelleted spores or individual organs), 2.5 μl of 10× Qiagen PCR buffer, 2.5 mM MgCl₂, 200 μM each deoxynucleoside triphosphate (dNTP) (Qiagen), 0.625 U Taq polymerase (Qiagen), and 0.5 μM each forward and reverse primer. PCR parameters for amplification were as follows: an initial DNA denaturation of 5 min at 95°C; 45 cycles of 1 min at 95°C, 1 min at 53°C, and 1 min at 72°C; and a final extension step at 72°C for 4 min. Amplification products (5 μl DNA) were electrophoresed on 1.1% agarose gels (1× Tris-borate-EDTA [TBE]), stained with ethidium bromide, and visualized under UV light. A commercial 100-bp ladder (Peqlab) was used as a size marker. For each PCR, positive (reference N. apis and N. ceranae DNA extracts) and negative (ddH₂O) controls were run along with DNA extracts of isolates as templates.

To differentiate between the species N. apis and N. ceranae, discriminating restriction endonuclease sites present in the PCR amplicon were used (33). The restriction endonuclease PacI provides one unique digestion site for N. ceranae, while the enzyme NdeI digests only N. apis. MspI digests both N. apis and N. ceranae and was used as a control for successful restriction digestion of PCR products.

The predicted restriction fragments produced from the digestion of the PCR amplicons are illustrated in Fig. S2 in the supplemental material. Amplicons were digested with MspI/PacI and with MspI/NdeI (New England Biolabs) in two reactions at 37°C for 3 h in order to analyze and confirm the presence of each Nosema species in each sample. Digestions were performed in a 12.5-μl volume with 7 μl of the amplified DNA and 1.5 U of each enzyme. For the reactions with MspI and NdeI, 1× NEBuffer 4 (provided by NEB with NdeI) was used as a buffer. For the reactions with MspI and PacI, 1× NEBuffer 1 plus bovine serum albumin (BSA) (provided by NEB with PacI) was used. Fragments were separated in a 3% NuSieve agarose gel (Cambrex Bio Science) in 1× TBE buffer with a 20-bp ladder as a size marker at 110 V for 1 h 30 min. Gels were stained with ethidium bromide and visualized under UV light.

Smear preparations for detection of developmental stages of Nosema spp.For the microscopic detection of developmental stages of Nosema spp. in smear preparations of bee tissue, adult bees infected with Nosema ceranae or Nosema apis were used. Brains, hypopharyngeal glands, malpighian tubules, fat bodies, and midguts were carefully isolated from individual bees as described above. Pieces of these tissues (area, 2 mm²) were placed on a microscopic slide for crush preparations using coverslips and the back of a pencil. After removal of the coverslips, the crushed tissue was air dried, fixed with 100% methanol, and air dried again prior to staining with Giemsa solution (1:10 Fluka Giemsa stain, modified solution) for 10 min. Stained tissues were rinsed with tap water, air dried, and embedded with Entellan (Merck). Microscopy analysis was performed at 1,000-fold magnification using a stereomicroscope (Leica).

In vitro germination of Nosema spores.Bee samples that tested positive for either N. apis or N. ceranae were used for isolation of the respective spores. The procedure for spore accumulation and purification was modified from the method of Chen et al. (13). Alimentary tracts of 20 individual bees were removed by snatching the sting with forceps and gently pulling the hindgut and the midgut out of the abdomen. The sting was cut with a scalpel, and the alimentary tract was transferred to a 1.5-ml reaction tube and crushed in a final volume of 1.5 ml sterile double-distilled water in a mixer mill (Retsch) with a 3-mm-diameter tungsten carbide bead (Qiagen) for 30 s at 30 Hz. The homogenate was filtered through a nylon cell strainer (Falcon) with a 100-μm mesh diameter. The filtrate was gently overlaid on a 90% Percoll (Sigma-Aldrich) cushion in a 15-ml reaction tube and was centrifuged twice at 15,000 × g for 45 min at 20°C in a Eppendorf 5810 R centrifuge using an F34-6-38 rotor. The small but dense band just above the bottom of the tube, which contained purified spores, was aspirated with a syringe and a Sterican 0.8-mm by 120-mm needle (Braun) and was transferred to a 15-ml reaction tube. Spores were washed three times with 8 volumes of double-distilled water and were centrifuged at 6,500 × g for 10 min at 20°C. After a final centrifugation step, the supernatant was removed, the pellet was resuspended in 500 μl AE buffer (Qiagen), and the spore concentration was determined using a hemocytometer. The identity of the spores was verified by PCR-RFLP as described above. Aliquots of purified N. apis or N. ceranae spores (20 μl; 2E+8 spores per ml) were spotted onto glass slides, air dried, and kept at different temperatures for different periods of time. Subsequently, germination was triggered by the addition of 30 μl of 0.1 M sucrose in PBS buffer to the air-dried spores (39), a procedure that mimics the natural conditions for the germination of environmental spores. Representative results obtained with freshly isolated, dried, and germinated spores (+22°C) or after storage of the dried spores at 4°C for 4 days are shown in Fig. 4. These experiments were repeated 10 times with different spore preparations (n = 10).

Data evaluation and statistical analysis.We compared surviving and collapsed colonies by using nonparametric chi-square tests, because the basal assumptions of parametric tests (i.e., normality and constant variance) were not satisfied. The distribution of colony losses differed between the years and between the seasons (see Tables 1 and 2); therefore, the data sets were analyzed separately. Chi-square tests were performed by comparing the infection statuses of the surviving colonies with those of the collapsed colonies. A P value of <0.05 was considered significant.

Incidences of Nosema apis and Nosema ceranae.During the entire period of our study, we observed the seasonality of Nosema-positive colonies that was described previously (20) (Fig. 1). The proportion of samples positive for Nosema spp. in the cohort monitored was always higher in the spring than in the autumn. In the spring, the proportion of Nosema-positive samples ranged from 22.4% (2007) to 35.4% (2008). In the autumn, the highest prevalence of Nosema spp. was detected in 2005 (12.7%) and the lowest in 2008 (5.2%).

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FIG. 1.

Epidemiological survey of 22 apiaries in the northeastern part of Germany for Nosema prevalence between spring 2005 and spring 2009. The prevalences of samples positive for Nosema spp. (filled diamonds), Nosema apis (open squares), Nosema ceranae (open triangles), and both species (mixed infections) (shaded circles) are shown at each time point. Diagnosis of Nosema spp. was performed microscopically. Nosema differentiation was performed via RFLP analysis of a PCR-amplified partial sequence of the 16S rRNA gene.

Since recent studies had suggested the replacement of N. apis by N. ceranae in Europe (33, 40), we expected to find hardly any N. apis-positive samples. Surprisingly, this was not the case. With the exception of spring 2007, N. apis was always more prevalent than N. ceranae. The proportion of N. apis-positive colonies in the spring ranged from 15.7% (2009) to 3.7% (2007), and that in the autumn ranged from 8.0% (2005) to 2.9% (2008) (Fig. 1). The proportion of colonies that tested positive for N. ceranae ranged from 14.9% (2007) to 4.1% (2005) in the spring and from 4.2% (2005) to 1.3% (2006) in the autumn (Fig. 1). In spring 2007, 14.9% N. ceranae-positive colonies and only 3.1% N. apis-positive colonies were detected in our cohort. A low prevalence of mixed infections with the same seasonal pattern (more positive colonies in the spring than in the autumn) was also detected throughout the study period (Fig. 1).

Colony losses during the study period.In order to evaluate the impact of Nosema infection on the mortality of honeybee colonies, we differentiated between colony losses in the summer season (weeks 15 to 35) and overwintering losses (weeks 36 to week 14 of the following year), and we related these to the detection of Nosema spp., N. apis, and N. ceranae in spring and autumn samples (Tables 1 and 2). The winter losses we observed in our cohort during the study period ranged from 22.4% (2005/2006) to 4.8% (2008/2009), showing that our study period covered winters with low to normal colony mortality rates as well as winters with rather high colony mortality rates (Table 2). We observed a similar variation, although on a much lower level (1.8% for 2007; 6.7% for 2008), for colony losses during the season (Table 1). Our data did not reveal a significant relation between the detection of Nosema infection in the spring and colony losses in the following season (Table 1, P values) or between detectable Nosema infections in the autumn and colony collapses during the following winter (Table 2, P values). In addition, no significant differences between the mortality rates of uninfected colonies and those of colonies infected by N. apis, N. ceranae, or both (mixed infections) could be established (P, ≫0.05).

Tissue tropism of N. apis and N. ceranae.While N. apis infections are reported to be restricted to the gut (16, 19, 21), N. ceranae could also be detected in hypopharyngeal glands, salivary glands, malpighian tubules, and fat bodies, indicating a more generalized infection (13), which could possibly be linked to the reported increased lethality of N. ceranae (27, 40). Since our results did not reveal the previously reported, and therefore expected, correlation between N. ceranae infection and colony losses (28, 37), we asked whether a less virulent form of N. ceranae, which did not cause such generalized infections, might be present in Germany. We therefore analyzed the tissue tropism of N. apis and N. ceranae in the infected bees collected in our study in detail but did not find any substantial differences from what had already been described (13). By the use of PCR analysis and RFLP differentiation, N. apis was detected only in the guts of infected bees, supporting the tissue tropism reported for N. apis (16, 17, 19). In contrast, using the same method, we detected N. ceranae in hypopharyngeal glands, brains, guts, malpighian tubules, and fat bodies (Fig. 2). For the majority of the bees, infection of the gut could be demonstrated. In addition to the detection of N. ceranae in the tissues, as reported by Chen and coworkers (13), we could detect N. ceranae in the brain as well. All N. ceranae-positive bees analyzed showed a rather generalized infection, with at least two different tissues testing positive for N. ceranae. However, using the less sensitive method of Giemsa-stained smear preparations, we could detect both N. apis and N. ceranae only in gut tissue (Fig. 3).

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FIG. 2.

Detection of Nosema spp. and differentiation via species-specific 16S rRNA gene RFLP in different tissues of infected bees sampled from colonies that tested positive for Nosema ceranae only. The tissues analyzed were hypopharyngeal glands (h. glands), brains, guts, malpighian tubules (m. tubules), and fat bodies.

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FIG. 3.

Detection of Nosema spp. via smear preparations in infected bees. Different organs of infected bees were analyzed for the presence of spores and vegetative stages of Nosema spp. by using Giemsa-stained smear preparations. A representative image from the gut of an N. apis-infected bee is shown. n, nucleus. Nuclei are stained red, and spores are stained blue-white.

Germination of N. apis and N. ceranae.Previous studies have shown that N. ceranae spores, but not N. apis spores, are sensitive to a temperature of −20°C (18). Since germination is the first step in the infection process, we analyzed the germination capacities of N. apis and N. ceranae spores exposed to low temperatures versus room temperature. We found that the germination of both Nosema species was affected after 4 days at 4°C. Estimation of the numbers of germinated spores and extruded polar tubes revealed that about 80% of the N. apis spores were still capable of germination, while N. ceranae spore germination was reduced to less than 10% (Fig. 4). In addition, the polar tubes extruded from the few N. ceranae spores still capable of germination were unusually short, most likely not representing true germination (Fig. 4).

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FIG. 4.

In vitro germination of N. apis and N. ceranae spores. Spores were isolated, air dried on glass slides, and kept at different temperatures for different periods. Representative results obtained after storage of the dried spores at +4°C for 4 days are shown in comparison with freshly isolated, dried, and germinated spores. Extruded polar tubes can be seen as curved lines in the images. White arrowheads point to extremely short, extruded polar tubes seen only for N. ceranae after 4 days at +4°C.