Open Veterinary Journal, (2026), Vol. 16(4): 2429-2439

Research Article

10.5455/OVJ.2026.v16.i4.44

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Microsporidia Nosema spp. of Apis mellifera were detected for the first time using microscope and molecular methods in the Iraqi Kurdistan region

Tishk H. Shekh Faraj^* and Rukhosh J. Rashed

Department of Horticulture, College of Agriculture Engineering Science, University of Sulaimani, Sulaymaniyah, Iraq

*Corresponding Author: Tishk H. Shekh Faraj. Department of Horticulture, College of Agriculture Engineering Science, University of Sulaimani, Sulaymaniyah, Iraq. Email: tishk.shekhfaraj [at] univsul.edu.iq

Submitted: 11/09/2025 Revised: 22/02/2026 Accepted: 07/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Nosemosis is considered an important disease causing a sudden decline in honeybees (Apis mellifera) worldwide, especially in the Iraqi Kurdistan region. Microsporidia have three recognized species (Vairimorphia ceranae, Vairimorpha apis, and Vairimorpha neumanni) on European bees globally and have not yet been comprehensively identified in the Iraqi Kurdistan region.

Aim: Due to the lack of extensive studies, including higher numbers of accessions and all three specific primers, to detect three recognized species (globally) of this parasite in the Kurdistan region of Iraq, the current study used microscope examinations and molecular methods [conventional polymerase chain reaction (PCR) analysis] for detecting Vairimorpha spp.

Methods: This study collected 51 samples taken at 17 locations in three seasons (Spring, Summer, and Fall) in the cities of Sulaymaniyah, Erbil, and Duhok. To conduct the study, two methods were applied to detect these parasites: a microscope (400× magnification) and molecular PCR (amplifying 16S rRNA gene).

Results: The sequenced PCR product gave the right size of V. ceranae only, and the blast was confirmed in the National Center for Biotechnology Information platform for the first time in the Iraqi Kurdistan region. According to traditional methods (microscopic examination), 15% (8 out of 51) of the accessions had Vairimorpha, compared with 50% according to molecular studies. Regarding the seasons, the microscope gave (29%, 5%, and 11%) Vairimorpha for spring, summer, and fall, respectively, compared with the molecular results (70%, 29%, and 52%).

Conclusion: Vairimorphia ceranae was the only species detected by amplification of the 16S rRNA amplicon. This species is widespread in wet months and at moderate temperatures.

Keywords: 16S rRNA gene expression, Microscopic examination, Vairimorpha species.

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Introduction

European honeybees (Apis mellifera L.) are important for the production of many valuable products, including honey, pollen, royal jell, and propolis (Alam et al., 2021). Moreover, honeybees play an important role in the pollination of many plant crops (Alemberhe and Gebremeskel, 2016).

Apis mellifera encountered different pathogens. Nosemosis, which has recently updated the nomenclature to (Vairimorpha) by Tokarev et al. (2020) due to genetic match with this new genus, is one of the most important diseases infecting the guts of adult workers (Goulson et al., 2015). This disease is caused by Vairimorpha, an obligate unicellular parasite of the microsporidia (Urbieta-Magro et al., 2019; Tokarev et al., 2020; Dong et al., 2021), which infects a wide range of vertebrates and invertebrates, including humans. They were discovered more than 160 years ago and include approximately 1,600 species (Murareanu et al., 2021).

Vairimorpha has many negative impacts on bee colonies. The first one is economic losses (Ütük et al., 2019) by decreasing honey production (Alam et al., 2021). In addition, the life span of adult workers decreased, and their mortality gradually increased in winter (Farag et al., 2024).

For decades, the disease of Vairimorpha was particularly described as a single species of Vairimorpha apis, which was initially detected in the European honeybee A. mellifera (Zander, 1909). In 1996, a new species of Vairimorpha was recognized for the first time in the Asian honey bee, Apis ceranae (Fries et al., 1996). In 2005, natural infection of Vairimorphia ceranae was reported in A. mellifera colonies from Taiwan in 2005 (Huang et al., 2007). Vairimorpha apis spores are oval in shape and approximately 6 × 3 µm in size (Zander and Bottcher, 1984). According to the description of Fries et al. (1996)the spores of V. ceranae are smaller and their size is around 4.7 × 2.7 µm. Chemurot et al. (2017) detected a new species Vairimorpha neumanni, in Uganda. The different symptoms presented by nosemosis in honey bees by these Vairimorpha species highlight the need to observe two different clinical types: nosemosis type A caused by V. apis and nosemosis type C caused by V. ceranae (Khezri et al., 2018). Vairimorpha apis is known to manifest as diarrhea (Fries and I, 1993), but this is not the case in V. ceranae (Paxton, 2010). However, some authors have reported that these two species have no clinical signs of infection (Higes et al., 2010a,b; Maiolino et al., 2014). The infection levels of these two Vairimorpha species differ between bee colonies (Chen et al., 2009; Mulholland et al., 2012; Chauzat et al., 2016; Martin-Hernandez et al., 2018). There were significant differences in morphology (Fries et al., 2013; Ptaszyńska et al., 2014), genome size (Chen et al., 2013; Gómez-Moracho et al., 2014), spore production (Huang and Solter, 2013;
Martin-Hernandez et al., 2018; Sinpoo et al., 2018), and virulence and impact on bee health (Paxton et al., 2007; Higes et al., 2008; Martin-Hernandez et al., 2011).

Under microscope examination, the spores of these two species morphologies are similar and cannot differentiate between V. apis and V. ceranae (Higes et al., 2010a,b). However, molecular methods have proven to be more effective than traditional methods in detecting Vairimorpha spp. Many polymerase chain reaction (PCR) protocols have been described, including PCR with specific primers (Chen and Huang, 2010), PCR-Restriction Fragment Length Polymorphism (Fries et al., 2006; Bourgeois et al., 2010), and real-time PCR (Cox-Foster et al., 2007; Martín-Hernández et al., 2007), or multiplex PCR (Sharifi and Mehrabadi, 2013).

Owing to the lack of comprehensive studies including higher numbers of accessions in the Kurdistan region of Iraq and all three specific primers to detect three recognized species (globally) of this parasite, the current study used microscope examinations and molecular methods (conventional PCR analysis) for detecting Vairimorpha spp.

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Materials and Methods

In 2024, a field survey was conducted in different locations of the Iraqi Kurdistan region, including Sulaymaniyah, Erbil, and Duhok cities, for spring, summer, and fall in 2024. In total, 51 samples were collected for all seasons Supplementary Table 1. For each sample, 50 honeybees were taken from the hive, sterilized with 70% ethanol, and frozen on the same day until testing. In addition, the climatic conditions were recorded during the sample collection, represented by measuring the temperature and humidity percentage.

Microscope examination

The research was conducted at the Horticulture Department of the Higher Education Laboratory of the University of Sulaimani/College of Agriculture Engineering Sciences, Sulaymaniyah, Kurdistan, Iraq. Using a normal hemocytometer is the simplest method for counting Vairimorpha spores. According to the protocol described by Human et al. (2013)the dilution factor was 1 ml: 1 bee. From each sample, 10 bees were taken, and their abdomens were separated with forceps and placed in a clean and sterilized petridish. Then, 10 ml of distilled water was added. The abdomens were crushed with a sterilized ceramic mortar and pestle until an aliquot was homogenized, then the mixture of solution was filtered through a 70 µM cell strainer to separate large particles that originated from the bees’ alimentary canals (Truong et al., 2021). One drop from the suspension was placed on a microscope slide, covered with a cover slip, and examined using a light microscope (LabMod/USA). Vairimorpha spores were seen at 400× magnification. Vairimorpha samples were counted using hemocytometer slides. The number of spores was taken from five squares of the hemocytometer (Raw spore count from 5 blocks • 4 million) / # of squares counted=number of spores per bee (Mortensen et al., 2020).

DNA extraction

Ten abdomens from each study location were separated from adult worker bees. The abdomens were homogenized using liquid nitrogen, from which 100 mg was used to extract DNA following the protocol of the DNA extraction kit (Biotech, Korea). The extracted DNA concentrations and purity were examined using Nano Drop (NanoPlus Uv/vis Spectrophtometer/Sweden). Regarding the contamination prevention steps, all the tips, 1.5 ml centrifuge tubes, and PCR tubes were sterilized under the conditions of 121°C and 15 psi for 15 minutes using an autoclave machine.

Polymerase chain reaction

For the PCR reaction, the final volume of 25 µl reaction mixture contains 5 µl of the DNA, 12.5 Master Mix (Sinaclon, Iran), 1.5 µl of the forward primer, 1.5 µl of the reverse primer, and 4.5 µl of ddH₂O. Thermocycle conditions of PCR following Chen et al. (2009). In brief, PCR amplification started with an initial denaturation at 94°C for 5 minutes, followed by two main stages. The first stage was for 10 cycles; 94°C for 15 seconds denaturation, annealing temperature 61.80°C for 30 seconds, and extension stage 72.00°C for 45 seconds. The second stage was for 20 cycles, including 94°C for 15 seconds denaturation, annealing temperature 61.80°C for 30 seconds, and extension stage 72.00°C for 45 seconds. This followed an additional extension step of 7 minutes at 72.00°C. The PCR products were run on 1% agarose gel in 1X TBA buffer treated with ethidium bromide and visualized under UV transillumination. To check the exact size of PCR products, a 1 kb Hyperlink ladder (Add - Bio, South Korea) was used. The PCR products of the six samples were sent for sequencing to the PharmaGene laboratory (Sulaymaniyah-Iraq). The products will be deposited in the GenBank database of the National Center for Biotechnology Information (NCBI).

Specific primers

Three sets of specific primers were ordered from the LIGO company for three species (Chen et al., 2008; Chemurot et al., 2017) (Table 1).

Table 1. Specific primers of all Vairimorpha species. Includes primer sequences, amplicon size, and annealing temperature.

Ethical approval

Not needed for this study.

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Results

A broad geographical survey was conducted in 2024, which resulted in the collection of 51 accessions from 17 different locations during three seasons (spring, summer, and fall) in 2024. All GPS data were uploaded into QGIS to draw the map (Fig. 1).

Fig. 1. Geographical distribution map of samples collected from four Iraqi cities. The collection sites are indicated with colored circles. QIGIS software was used to draw the map.

The laboratory investigation of adult honeybees (A. mellifera) was conducted to detect their infection with Vairimorpha species using both microscopic and molecular methods. This delineated the results that out of 51 colonies from all three seasons, 8 and 26 colonies were infected, respectively.

To examine the presence or absence of pathogen spores in the initial step, a microscopic method was used at 400× magnification. In total, 8 of 51 colonies were detected in all three studied seasons. These results explore that the pathogen was found in 29% of the locations studied in Spring, including Piramagroon, Raparin, Sulaymaniyah/College of Agricultural Engineering Sciences, Penjwen, and Sharbazher. In Summer, this result decreased to 5% and was found only in Sharbazher. Regarding the fall season, the current study noticed that the infections with this pathogen slightly increased to 11% of the studied locations (Raparin and Sulaymaniyah/College of Agricultural Engineering Sciences) (Fig. 2).

Fig. 2. Vairimorpha spores observed during counting in 5 of the 16 observation fields of a hemocytometer. Observations were performed at 400× magnification under a light microscope. The real concentration of the spore solution was calculated based on the spore count.

Hemocytometer results showed that the maximum number of spores was found in the Piramagroon (7.5 × 10⁵) in the spring, whereas in summer, only detected in Sharbazher (1.75 × 10⁶). The maximum number of falls was recorded in Sulaymaniyah/College of Agricultural Engineering Sciences (6.5 × 10⁶) (Table 2).

Table 2. Vairimorpha ceranae spores counted by a hemocytometer (Mortensen et al., 2020), locations, and seasons.

In Iraq, the current study is the first attempt to use three specific primers belonging to all three species (V. ceranae, V. apis, and V. neumanni). The aim of using three specific primers is to determine how broadly these nosemosis disperse among apiaries in Iraqi Kurdistan region, as many new bee strains have been imported from neighboring countries.

The DNA amplifications of the 51 studied samples were conducted using all specific primers. Vairimorphia ceranae was the only species visualized on agarose gel and gave the exact band of 218 bp (Fig. 3).

Fig. 3. Gel electrophoresis of PCR productions using specific primers 16S rRNA gene. L=1 Kb Hyperlink Ladder; N=negative control; P=positive control; bp=base pair.

To confirm this result, the PCR products of six samples were sent for sequencing at the Pharmagene Laboratory (Iraq/Sulaimaniyah). These sequences were blasted against the GenBank database (NCBI) using nucleotide Basic Local Alignment Search Tool (BLAST). The sequences of our V. creanae isolates were high-percentage identical to the sequences previously deposited in GenBank (Table 3). These were submitted to the NCBI GenBank with accession numbers PX760833, PX760834, PX760835, PX760836, PX760837, and PX760838, respectively. The current study sequences (numbers 1–6) were intermingled with strains downloaded from the NCBI database
of V. ceranae (seven accessions starting from 7 to 13), V. apis (three accessions starting from 14 to 16), and V. neumanni (one accession number 17) (Fig.4). This alignment clearly illustrates that our sequences are aligned with published sequences of V. ceranae from the NCBI database. Vairimorpha. apis and Vairimorpha neumanni sequences are aligned and clearly separated.

Table 3. Results of top-hit accessions from NCBI with the study sequenced PCR products.

Fig. 4. The study alignment explains the similarities and differences between our SSU16s rRNA gene sequences of V. ceranae and the NCBI sequences of V. ceranae and V. apis, respectively. DNA nucleobase: (A)=Adenine; (C)=Cytosine; (G)=Guanine; (T)=Thymine.

Molecular PCR results for all studied sites show that 26 locations have exact bands for V. ceranae are 12, 5, and 9 in spring, summer, and fall, respectively (Table 4). Regarding the chi-square test results, no significant differences among seasons were found (Table 5). In addition, the comparison between microscope and molecular methods, the significant χ² result supports that PCR is more sensitive than microscopy (Table 6).

Table 4. Molecular study using PCR shows the number of positive bands for all locations in different seasons.

Table 5. Molecular study using PCR showing the number of positive and negative bands for all locations in different seasons using χ2 test.

Table 6. Comparison of microscope and molecular methods for detecting Vairimorpha spp. using χ2 test.

According to the seasons, the highest number of samples with the exact band was found in spring (70%), and in summer and fall (29%) and (52%), respectively (Fig. 5).

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Discussion

Vairimorpha is one of the most important diseases affecting adult honeybees worldwide and has an economic impact on beekeeping (Khezri et al., 2018). Vairimorpha sp. is considered a hidden pathogen in the honeybee hive, especially in the early stages. For instance, V. ceranae in bee colonies revealed no clear symptoms at the initial stage and cannot be seen until the hive reaches the final level of decline (Nabian et al., 2011). Therefore, the current study used two different methods, microscopic and conventional PCR, to investigate the samples collected from various apiaries in the Iraqi Kurdistan region. Clear visualizations of these pathogens are needed to identify the most effective and environmentally friendly means of control.

According to previous studies, the molecular mean is more sensitive than the traditional microscopic examination method in detecting this pathogen (Webster et al., 2008). This statement agrees with the results of the current study, in which out of 51 examined colonies using both methods, 34 colonies were infected with the Vairimorpha pathogen, of which 8 colonies and 26 colonies were detected by microscopic and molecular methods, respectively.

According to Kidoud et al. (2023) climate is an effective factor in increasing the prevalence of Vairimorpha species. This is congruent with the results of the current study (high in spring), which may refer to the high level of humidity in spring (Alam et al., 2021), and the infections were increased dramatically when the temperatures ranged between 25°C and 37°C and in wet months (Kareem et al., 2021; Manjy and Shaher, 2025). Researchers have also confirmed that the abundance of nutrition in the spring season is another factor that promotes the prevalence of Vairimorpha spores (Degrandi-Hoffman et al., 2024).

According to previous studies on pathogen nosemosis, the molecular method considered most accurate to the microscope mean in detecting different species of Vairimorpha (Michalczyk et al., 2011). The traditional method is unable to detect Vairimorpha spores when their concentrations in a bee’s gut are below 2.4 10⁴ spores per gut (Truong et al., 2021). Several molecular studies have been conducted worldwide, and V. ceranae and V. apis have been detected (Alvarez-Ramos et al., 2013; Tokarev et al., 2018; Moeini et al., 2022; Farag et al., 2024). Chemurot et al. (2017) detected a new species on A. mellifera named V. neumanni in Uganda.

This study confirmed that V. ceranae was the only species found to infect honeybees from the apiaries in all the areas under study. The reasons for this are related to the adaptations of V. apis with A. mellifera, as V. ceranae is newly transferred to European honeybees (Utuk et al., 2016).

Our PCR products visualized on agarose gel (1%) gave a band of 218 bp according to the DNA Ladder (1 kb Plus DNA Ladder) and GenBank BLAST, which is in accordance with Chen et al. (2008)and Chen et al. (2009). The current study revealed that 49% (26 out of 51) of the samples produced bands of the correct size. This is higher than the accuracy of the PCR methods in detecting these pathogens (Michalczyk et al., 2011). The percentage of the seasonal results from both methods in the comparison charts in (Fig. 5) illustrates the same pattern of obligated fungi spores, which confirms the previous statements.

Fig. 5. Comparison between the percentage results of microscope examination and PCR of Vairimorpha spores in spring, summer, and fall.

A few limitations should be considered. Initially, the sample size was restricted because additional samples were not consistently available during the sampling period, which may reduce statistical power and limit generalization to all apiaries in the Iraqi Kurdistan region. Second, quantitative PCR (qPCR) was not used because it was unavailable in our laboratory; therefore, we relied on conventional PCR, which was adequate for presence/absence confirmation because a substantial proportion of samples (~50%) showed the expected amplicon band. However, the lack of qPCR prevented quantification of infection intensity (pathogen load) and may have reduced sensitivity for very low-level infections; future studies should include larger sampling and incorporate qPCR (and ideally sequencing) for stronger inference.

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Conclusion

In the present study, V. ceranae was the only species detected through amplification of the 16S rRNA gene, which represents the first research conducted in the Kurdistan Region of Iraq. This exclusive presence may refer to the exist of suitable environmental conditions on one hand, and on the other hand, it refers to the absence of solid management and ignorance by the beekeepers. This study proved that this species was widely distributed during wet months with moderate temperatures and showed the highest prevalence in spring across all three cities, Sulaymaniyah, Erbil, and Duhok. This pathogen causes severe but hidden damage to adult worker bees, leading to colony decline, shortened lifespan, and pollination activity disruption. Therefore, beekeepers should manage their apiaries at appropriate times to prevent the spread and prevalence of this disease. However, the limited number of samples collected and the use of only a single gene display major limitations of this study, restricting its scope to a small geographical area rather than the entire Iraq. Accordingly, to obtain more accurate and comprehensive results, future studies should include a larger number of sampling locations and apiaries, employ multiple genetic markers, and sequence a higher number of PCR products.

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Acknowledgment

The authors would like to express gratitude to the University of Sulaimani, College of Agriculture Engineering Sciences, Department of Horticulture in Sulaymaniyah city, beekeepers, and friends for their assistance in collecting bee accessions in all 17 locations.

Conflicts of interest

The authors have no conflicts of interest to declare.

Funding

No funding is available for this project.

Authors’ contribution

(T.H.S.F. and R.J.R.) T.H.S.F. and R.J.R.: Design the study. T.H.S.F.: Methodology, sample collection, and writing stage of the manuscript. R.J.R.: Revised the manuscript. The final manuscript has been read, corrected, and approved by all authors.

Data availability

This article contains all the data created or examined in this investigation.

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