Molecular Detection of Nosema spp. in Three Eco Regions of Slovakia

Microsporidia are unicellular obligate intracellular parasitic fungi that infect a wide range of vertebrates and invertebrates. There are two known species of microsporidia infecting honey bees in Slovakia- first Nosema apis and also Nosema ceranae. Our aim was to examine samples of honey bees collected from bee queen breeders in three ecoregions of the Slovak Republic in 2021 and 2022. First, microscopic diagnostics were used, and then randomly selected samples were examined using molecular methods. There were 4018 samples examined using microscopic diagnostics and the positivity was demonstrated in 922 samples. From the microscopically diagnosed positive samples, 507 samples were randomly selected, and using molecular methods, the positivity was proved in 488 samples. After sequencing the positive PCR products and comparing the sequences (BLAST) with the sequences stored in the gene bank, the Nosema ceranae species was detected in all positive samples.


Introduction
Microsporidia are unicellular [1] obligate intracellular parasites [2], parasitic fungi [3] that infect a wide range of vertebrates and invertebrates, including humans. They were discovered more than 160 years ago and include around 1600 species [4].
These parasitic fungi are eukaryotes without typical features of eukaryotes such as mitochondria, peroxisomes, and Golgi apparatus. Outside the body of their host, they exist in the form of highly resistant chitin-coated infectious spores, ranging in size from 1 to 40 µm, and may occur in water or soil [1,2]. They can be viable in the environment for over a year [5]. There are three known species of microsporidia infecting honey bees: Nosema apis, Nosema ceranae, and Nosema neumanni.
Nosema apis has been known to science for more than 100 years [2], since 1909, in the honey bee-Apis mellifera. In 1996, Nosema ceranae was first identified in the Asian honey bee, Apis cerana, and in 2017, Nosema neumanni was discovered as a new species of this parasite, which is found, so far, only in Uganda [6].
In local geographical conditions, Nosema apis was the first to occur, Nosema ceranae occurred later [7], often appearing as a co-infection. Until 2008, Nosema apis was the only diagnosed species of the microsporidian parasite in Slovak honey bees causing diarrhea [8]. This disease is often associated with CCD-colony collapse disorder [9].
Nosematosis causes physiological and behavioral changes in honey bees [3]. Clinical signs begin to appear only after the spores multiply in the epithelium of the digestive system [10]. During Nosema apis infection, one may easily notice the bee's fecal material on the honey frames and in the area of the hive, and crawling honey bees around the hive [7]. microscope at 400× magnification in a drop of crushed bee abdomens mixed with a physiological solution [13].
Nosema spores have a regular shape, size, and so-called refraction of light under the microscope. Nosema apis may be distinguished from Nosema ceranae by the size of the spores. The spores of Nosema apis have a shape between oval to barrel; they are from 4.9 to 6.9 µm long and 2.7 to 3.9 µm wide. Nosema ceranae has smaller, cylindrical spores with a pointed end measuring from 3.9 to 5.4 µm in length and 2.0 to 2.9 µm in width [20].
To quantify spore counts, a hemocytometer count is used, where we count the number of spores in all squares of the hemocytometer and, after plugging into the formula, calculate the number of spores per bee [21]. To better detect spores, they can be stained by Giemsa staining with a commercially available dye; this method is used to distinguish Nosema spp. spores from other microbes in the sample [22], or we can use toluidine staining [21]. Using fluorescence microscopy, we can distinguish live spores from dead spores of Nosema cerenae in addition to detecting infection.
Among microscopic diagnostics, transmission electron microscopy-TEM-is the gold standard in identifying microspores, which helps to distinguish Nosema spp. based on the observation of the number of their polar filaments in the sporoplasm.
The histopathological method is a very sensitive and specific method for the detection of nosematosis and has the advantage of being able to provide a diagnosis in colonies without signs of disease [23].
Microscopic quantification and detection of Nosema spores is a reliable method of detection. However, differential diagnosis is not performed microscopically, except by TEM, which is time-consuming and also expensive [13]. Therefore, the polymerase chain reaction method was developed as a tool to diagnose the species [5]. PCR is a very sensitive method that enables the detection of the parasite even at very low levels of infection and may identify all stages of the microsporidia's life cycle [21]. In particular, multiplex PCR, PCR-RFLP, and qPCR are used [24]. The first step of the reaction is the isolation of DNA from the samples, following a tissue protocol using commercially available kits [5]. This extracted DNA is the template for the preparation of the PCR mix to which special commercially available primers for the detection of Nosema spp. are added [25]. The use of primers designed based on the 16S rRNA minor subunit gene is recommended as the most reliable method. DNA amplification is performed on a thermocycler, followed by the visualization of PCR products on agarose gels. The resulting PCR reaction products can then be sent for sequencing, where the exact sequence is harvested, and the result is compared to the sequences in the Gene Bank [13,21].
In addition to the aforementioned types of PCR, diagnosis using the LAMP method is also possible, during which both Nosema spp. can be detected. This method is based on the isothermal amplification of DNA fragments and the authors report that it is highly specific and even more sensitive than PCR alone [26,27].
In field settings, the immunodiffusion serological method can be used, which is mainly suitable for the detection of Nosema cerana [28].
All methodologies for the diagnosis of Nosema spp. are described in the BEEBOOK practical manual of standard methods for the honey bee [21].
The aim of this pilot study is to investigate the incidence of Nosema spp. in three ecoregions of Slovakia in 2021 and 2022 by comparing our results with those of other countries that have performed species diagnostics in their territory and monitored the prevalence of Nosema spp., and to compare the accuracy of the microscopic diagnoses with molecular methods.

Sampling
Samples of dead honey bee workers for the detection of Nosemosis and Acarapidosis were collected by the bee queen breeders in the breeding farms from bottom hive debris. Sampling of dead honey bee workers for the examination was performed at the beginning of the year in January and February. The beekeepers sent the samples, whose number is specified to be 30-50 bodies per bee colony, to the Institute of Apiculture Liptovský Hrádok.
In 2021, 39 bee queen breeders were registered in Slovakia and a total of 2028 samples were sent for examination. In 2022, 32 bee queen breeders were registered and a total of 1990 samples were sent for testing. The number of keepers from each part of the country is given in Table 1. The abdomens of 50 dead winter honey bees were cut from their bodies. Then, the abdomens were crushed using a mortar and pestle in distilled water.
After the bee abdomens were crushed with a mortar and pestle, the solid part was separated from the liquid part. A drop of the liquid part was placed on the glass slide and covered with a coverslip. The preparation was viewed under a microscope with phase contrast 400× magnification, as it helps distinguish Nosema spores from yeast or other particles. The Nosema spores were observed under the microscope from several fields of view, and then the results of the number of spores were averaged and evaluated. In this case, 0 spores viewed were evaluated as negative samples, 1-19 spores in the field of view were determined as + weak Nosema infection, 20-100 spores in the field of view were identified as ++ moderate Nosema infection, and above 100 spores in the field of view were evaluated as +++ strong Nosema infection. The liquid part of the samples, which was evaluated under the microscope as positive for Nosema spp., was labeled in test tubes, frozen, and sent for molecular diagnosis and detection of Nosema spp. to the University of Veterinary Medicine and Pharmacy in Košice.

PCR-Based Diagnostics of Nosema spp.
The frozen samples of the liquid component of honey bee abdomens were thawed at room temperature. In order to isolate DNA from the samples, DNA-sorb-AM (AmpliSens, Moscow, Russia) kit for the extraction and purification of nucleic acids from tissues was used according to manufacturer's instructions. Then, 0.5 mm and 1.0 mm zirconium beads were added to each sample in order to break hard coat of Nosema spores; PRECELLYS 24 (Bertin, MD, USA) tissue homogenizer was used, and the device was set to 6500 rpm and 2 × 45 s.

Reaction Mixture Preparation
A duplex PCR protocol by [29] was used for the preparation of the reaction mixture, which was modified as follows: 2.5 µL buffer, 2 µL MgCl 2 , 0.5 µL dNTPs, 0.3 µL DNA polymerase (FIREPol DNA Polymerase produced by Solis BioDyne, Tarfu, Estonia), and was used for a single sample. Then, we proceeded as instructed in their protocol. In order to diagnose species of Nosema spp., two 30 picomole primers were added to the reaction mixture, 0.6 µL to each (Nosema apis for the amplification of 321 bp) APIS FOR-5 -GGGGCCATGTCTTTGACGTACTATGTA-3 and 321 APIS REV 5 -GGGGGGCGTTTA AAATGTGAAACAACTATG-3 and (Nosema ceranae for the amplification of 218 bp) MITOC FOR-5 CGGCGACGATGTGATATGAAAATATTAA-3 and 218 MITOC REV 5 -CCCGGTCA TTCTCAAACAAAAAAACCG-3. 2.5 µL of DNA template was added to the mixture, and PCR water was filled to final volume of 25 µL.
PCR was performed using a VWR RISTRETTO Personal Thermocycler according to the protocol of Ostroverkhová et al., 2020. ELFO was performed using 1.5% agarose gel after PCR reaction had been completed. After assessing the results using a UV Transilluminator, the DNA concentration was measured using the NanoDrop, and the PCR products were sent for sequencing. The obtained sequences were compared with the sequences stored in the gene bank using BLAST program. https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 20 November 2022).

Statistical Analysis
We performed statistical analyses of our results. To obtain a representative value, we calculated the mean, followed by the median, minimum value, maximum value, and coefficient of determination. To see if there is a linear dependence between our numbers of positive samples in both years and also the numbers of negative samples, we calculated the correlation coefficient between the positive and negative samples using the coefficient of determination-R 2 Statistical analyses were performed using the Microsoft Excel software package. To calculate the correlation from the calculated value of determination, we used the formula for calculating the correlation coefficient: R = √ (R 2 ).

PCR Diagnostics of Nosema spp.
All samples, which underwent DNA isolation and subsequent PCR amplification using primers for Nosema apis (amplicon 321 bp) and Nosema ceranae (amplicon 218 bp) after the application of an agarose gel and consecutive electrophoresis using UV Transilluminator, were visualized and determined as being positive for Nosema ceranae ( Figure 1).
PCR was performed using a VWR RISTRETTO Personal Thermocycler according t the protocol of Ostroverkhová et al., 2020. ELFO was performed using 1.5% agarose g after PCR reaction had been completed. After assessing the results using a UV Transillu minator, the DNA concentration was measured using the NanoDrop, and the PCR prod ucts were sent for sequencing. The obtained sequences were compared with the sequence stored in the gene bank using BLAST program. https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 20. November 2022).

Statistical Analysis
We performed statistical analyses of our results. To obtain a representative value, w calculated the mean, followed by the median, minimum value, maximum value, and co efficient of determination. To see if there is a linear dependence between our numbers o positive samples in both years and also the numbers of negative samples, we calculate the correlation coefficient between the positive and negative samples using the coefficien of determination₋R 2 Statistical analyses were performed using the Microsoft Excel software package. T calculate the correlation from the calculated value of determination, we used the formu for calculating the correlation coefficient: R = √(R²).

PCR Diagnostics of Nosema spp.
All samples, which underwent DNA isolation and subsequent PCR amplification u ing primers for Nosema apis (amplicon 321 bp) and Nosema ceranae (amplicon 218 bp) afte the application of an agarose gel and consecutive electrophoresis using UV Transillum nator, were visualized and determined as being positive for Nosema ceranae (Figure 1).  Subsequently, out of 470 positive microscopically examined samples, 81 samples of crushed honey bee abdomens were randomly selected and examined using molecular methods in order to confirm and identify species of Nosema spp. in Slovak honey bee colonies.

Evaluation of the Results of Examined Samples in 2022 in Three Ecoregions of Slovakia
In 2022, there were 1990 microscopically examined samples of dead honey bees collected from bee queen breeders in Slovakia. In total, 915 samples were examined from the western ecoregion, while 211 (23.06%) were positive and 704 (76.94%) were negative for  Subsequently, out of 470 positive microscopically examined samples, 81 samples of crushed honey bee abdomens were randomly selected and examined using molecular methods in order to confirm and identify species of Nosema spp. in Slovak honey bee colonies.

Evaluation of the Results of Examined Samples in 2022 in Three Ecoregions of Slovakia
In 2022, there were 1990 microscopically examined samples of dead honey bees collected from bee queen breeders in Slovakia. In total, 915 samples were examined from the western ecoregion, while 211 (23.06%) were positive and 704 (76.94%) were negative for Nosema spp. Next, there were 568 samples examined from the central ecoregion-129 (22.71%) were positive and 439 (77.29%) were negative. From the eastern ecoregion, 507 samples were examined, where 112 (22.09%) were positive and 395 (77.91%) were negative. The total balance of samples examined by the microscopic method was 452 positive and 1538 negative samples ( Figure 2).
Consecutively, whereas there were 452 positive microscopically examined samples in total, 426 samples of crushed honey bee abdomens were randomly selected and examined using molecular methods to confirm and specify Nosema spp. species in honey bee colonies in Slovakia. From the area of the western ecoregion of the country, 195 samples were examined-there were 193 (98.97%) positive samples and 2 (1.03%) negative samples. Then, there were 123 samples from the middle ecoregion-118 (95.93%) tested positive and 5 (4.07%) negative. In the case of the eastern ecoregion, 108 samples were examined-104 (96.30%) were positive and 4 (2.58%) negative samples. The total balance of samples examined by molecular methods was 415 (97.42%) positive and 11 (2.58%) negative samples ( Table 2).

Comparison of the Number of Positive Cases in Three Slovak Ecoregions from Samples Collected in 2021 and 2022
In Taking into account the fact that the number of examined samples was almost the same, it indicates that there were 13 more positive samples in 2021. The most significant contrast between the numbers of examined samples was present in the eastern ecoregion of Slovakia. During two years, 1107 samples were examined; there were 93 more samples in 2021, and the positivity was determined in 34 more samples in comparison with 2022 ( Figure 3).

Evaluation of Statistical Analysis of Results
The tests and analyses performed allowed us to determine the parameters described in Table 3.
The mean, the median, the minimum and maximum values, the coefficient of determination-R 2 , and the correlation coefficient-R were calculated from the R 2 value.

Evaluation of Statistical Analysis of Results
The tests and analyses performed allowed us to determine the parameters described in Table 3.   The correlation coefficient for positive and negative microscopy samples for 2021, R = 0.7785, indicates to us a moderately strong dependence, and we rate the correlation coefficient for positive and negative microscopy samples for 2022, R = 0.9871, as a particularly strong correlation coefficient.
We rate the correlation coefficient for positive 2021 molecular diagnostics samples, R = 0.8950, and the correlation coefficient for positive 2022 samples, R = 0.8064, as a particularly strong correlation coefficient.
We rate the correlation coefficient for negative molecular diagnostic samples for the year 2021, R = 0.8064, and the correlation coefficient for negative samples for the year 2022, R = 0.8950, as a particularly strong correlation coefficient Table 3.
From these analyses, we can conclude that there is a direct positive correlation and a direct linear dependence between the values of both the positive and negative samples for both 2021 and 2022 (Figures 4 and 5). mination-R2, and the correlation coefficient-R were calculated from the R2 value.
The correlation coefficient for positive and negative microscopy samples for 2021, R = 0.7785, indicates to us a moderately strong dependence, and we rate the correlation coefficient for positive and negative microscopy samples for 2022, R = 0.9871, as a particularly strong correlation coefficient.
We rate the correlation coefficient for positive 2021 molecular diagnostics samples, R = 0.8950, and the correlation coefficient for positive 2022 samples, R = 0.8064, as a particularly strong correlation coefficient.
We rate the correlation coefficient for negative molecular diagnostic samples for the year 2021, R = 0.8064, and the correlation coefficient for negative samples for the year 2022, R = 0.8950, as a particularly strong correlation coefficient Table 3.
From these analyses, we can conclude that there is a direct positive correlation and a direct linear dependence between the values of both the positive and negative samples for both 2021 and 2022 (Figure 4 and Figure 5).

Discussion
The present pilot study was carried out in 2021 and 2022. The aim of the study was to research the incidence of Nosema spp. species in Slovakia,  mination-R2, and the correlation coefficient-R were calculated from the R2 value.
The correlation coefficient for positive and negative microscopy samples for 2021, R = 0.7785, indicates to us a moderately strong dependence, and we rate the correlation coefficient for positive and negative microscopy samples for 2022, R = 0.9871, as a particularly strong correlation coefficient.
We rate the correlation coefficient for positive 2021 molecular diagnostics samples, R = 0.8950, and the correlation coefficient for positive 2022 samples, R = 0.8064, as a particularly strong correlation coefficient.
We rate the correlation coefficient for negative molecular diagnostic samples for the year 2021, R = 0.8064, and the correlation coefficient for negative samples for the year 2022, R = 0.8950, as a particularly strong correlation coefficient Table 3.
From these analyses, we can conclude that there is a direct positive correlation and a direct linear dependence between the values of both the positive and negative samples for both 2021 and 2022 (Figure 4 and Figure 5).

Discussion
The present pilot study was carried out in 2021 and 2022. The aim of the study was to research the incidence of Nosema spp. species in Slovakia,

Discussion
The present pilot study was carried out in 2021 and 2022. The aim of the study was to research the incidence of Nosema spp. species in Slovakia, The identification of Nosema spp. in the samples was first examined by microscopic methods. Subsequently, diagnosis was performed using molecular methods that can not only detect but also determine the species of the pathogen [30]. In all 488 positive samples examined by molecular methods, Nosema ceranae species was detected, whose size corresponds to 218 bp [31], as presented in (Figure 1) above. Due to the high sensitivity of molecular methods, it was possible to demonstrate more accurately which samples were positive and which were negative in samples previously identified as positive using the microscopic method. The determination of false-positive samples by light microscopy over two years ranged from 2 to 8%. Statistical analyses of the results showed a strong positive linear relationship between the variables studied. We also observe a positive correlation between the number of bee queen breeders (Table 1) in 2021 and 2022 with the number of  samples analyzed by microscopic methods (Table 2).
The last study of the incidence of mono and co-infections of the Nosema apis and Nosema ceranae species was conducted in Slovakia in 2009 and 2010 when Nosema ceranae was diagnosed in more than 90% of the samples. There was a recorded decrease in the incidence of Nosema apis and an increase in the incidence of Nosema ceranae in Slovakia [8]. Based on the latest data of the most recent (and ongoing) research that took place in 2021, Nosema ceranae was confirmed in all examined samples at the authors' workplace [32].
This fact may be related to the gradual displacement of Nosema apis by Nosema ceranae from milder climatic conditions. An increasing proportion of colonies infected with Nosema ceranae has been documented in many regions of the world in comparison to Nosema apis. The increased number of co-infections suggests that Nosema ceranae is moving from southern mild climates to northern subtropical climate regions and the Nosema apis is gradually being displaced [33]. The climate could be an important factor explaining the differences in the species distribution of this parasite.
In Sweden, Nosema apis is still prevalent in 80% of cases and 17% are co-infections. Co-infections prevail in Scotland, Argentina, and Australia. In Serbia and neighboring countries such as Croatia, Bosnia and Herzegovina, Montenegro, and North Macedonia, Nosema ceranae dominates. Furthermore, only one species-Nosema ceranae-may be found in Greece, Italy, eastern Azerbaijan, and Saudi Arabia [34]. Nosema ceranae displaces Nosema apis in the Apis mellifera population [35]. It is assumed that Nosema ceranae is the cause of increased colony losses registered in Europe and the United States [33]. In Spain, Nosema infection is even associated with reduced honey production and increased mortality. In recent years, the infection of honey bees caused by Nosema spp. has been reported in many European countries: Spain, France, Greece, Italy, Germany, Switzerland, Denmark, Finland, Hungary, Slovenia, and Bosna and Herzegovina.
Nosema ceranae was primarily present in most of the investigated honey bee colonies from honey bee societies of infected European countries. In Italy, Nosema ceranae is definitely widespread and has replaced Nosema apis.
The world trade in honey bee products and materials, and, especially, the trade in bee queens, may be a source of infection in some regions [34].
According to recent research in Bulgaria, where honey bee honey was used as a sample for DNA pathogen diagnostics, an increased prevalence of Nosema ceranae over Nosema apis was demonstrated. This points to the fact that not only the bee but also bee products such as honey can be a powerful resource for effective biomonitoring of current honey bee diseases [36].
Due to the increasing number of positive samples for Nosema ceranae in Europe and the world as well, which is devastating for honey bee colonies, it is necessary to continue to investigate the incidence of the species, possible mono or co-infections with Nosema spp., the increase in positive samples, and possible causes of the emergence and development of the infection. Last, but not least, it is important to research the possibilities of early diagnosis of Nosema ceranae infection and the prospect of treatment.

Conclusions
Our results confirm that Nosema ceranae is present throughout Slovakia in all three ecoregions. This is probably related to the gradual displacement of Nosema apis by Nosema ceranae worldwide. We have confirmed that molecular methods are very sensitive methods for the detection of Nosema spp. and have the advantage of being able to determine the species of the pathogen under investigation.