RESEARCH ARTICLE J Res Vet Med. 2022: 41 (2) 67-69 DOI:10.30782/jrvm.1121263 Prevalence of Nosema ceranae in North and South Regions of Azerbaijan Ahmet Onur GIRISGIN1, Rafiga GAZİ2, Barat AHMEDOV2 , Armağan Erdem ÜTÜK3, Levent AYDIN1 1 Bursa Uludag University, Faculty of Veterinary Medicine, Department of Parasitology, Bursa, Turkey 2 Institute of Zoology of Azerbaijan National Academy of Science, Center of Applied Zoology, Baku, Azerbaijan 3 Çukurova University, Faculty of Veterinary Medicine, Department of Parasitology, Adana, Turkey Received 30-05-2022 Accepted 03-10-2022 Abstract Nosemosis is one of the most dangerous infectious diseases of honeybees in Azerbaijan and the world. Nosema ceranae is the dominant spe- cies in Azerbaijan, and this study aimed to detect the prevalence of the infection in the country. For this purpose, an average of 100 honeybee samples were collected from 64 hives, 24 from three regions in the north and 40 from five areas in the south. In the lab, the abdomens of 50 bees from each group were dissected and crushed in a container, adding 50 ml of distilled water. According to obtained data after microscopic examination, the N. ceranae spores were found to have a high-level prevalence in northern regions (45.8% average) than in the southern areas (22.5% average) in Azerbaijan. Molecular diagnoses of Nosema-positive samples have been performed with PCR and N. ceranae has been detected from all regions. Data show us that the Nosemosis is common in Azerbaijan and is a significant threat in the beekeeping industry. Keywords: Nosema ceranae, prevalence, honeybee, Azerbaijan Introduction A. mellifera by N. ceranae clearly showed that this parasite was highly pathogenic to its new host2. It induced signifi- Beekeeping is one of the important economic sectors in the cantly higher bee mortality than N. apis8. world, as well as in Azerbaijan. In recent years, the country has been making significant progress with approximately The existence of Nosema apis in Azerbaijan has been not- 500.000 colonies and annual honey production of 6.000 ed in many scientific publications and conferences, and tons1. reported one the most prevalent diseases among the hon- eybees9,10. Recently, the existence of N. ceranae has been The productivity of the bees depends on both physiological recorded in our preliminary research before11. characteristics and health levels. In case the disease occurs in the bee colonies, the functional ability of the bees falls, This study aimed to determine the prevalence of N. ceranae and the losses become happening in the bee colonies. No- infection of honeybees in southern and northern regions sema is one of the microsporidian species and is becoming in Azerbaijan. a dangerous honeybee pathogen with its two species: Nose- ma apis and N. ceranae2,3. These parasites infect the diges- Material and Method tive system of adult bees and cause significant bee losses The study was conducted in May-June 2019. Since hon- worldwide4-7. The first reported experimental infection of eybees are invertebrates, there was no need for an ethics * Corresponding author: Ahmet Onur GIRISGIN, Tel: 02242941317, aogirisgin@uludag.edu.tr 67 Girişgin et al. 2022 committee report. An average of 100 worker bee samples per hive was collected from three regions in the north of Azerbaijan (Guba, Gusar, Shabran) and five in the south (Astara, Lankaran, Masalli, Jalilabad, Bilasuvar). Samples were collected from 64 hives, eight from each region in different places. The samples were put into specific plastic boxes and were brought to the laboratory to keep cold. To determine the Nosema spores in the sampled bees, 50 bees were dissected from each group. The abdominal parts of the bees were cut and crumbled by adding 50 ml of dis- tilled water in a large tube. After the resulting suspension was filtered through the filter, 0.1 ml of liquid was taken, dripped on a hemocytometric slide, covered with a cover Figure 1. The location of the Azerbaijani regions in the map from glass and examined under an ×400 magnified light micro- which the samples were taken. scope. The total number of spores (N) per bee is found with this formula: N= S×4×106 / 80; where ‘S’ is the total spores Nowadays, infection with N. ceranae has been proved al- in five cytometric areas on the slide12,13. The average num- most on all continents. Recent reports have confirmed ber of spores was determined in these areas, and the total changes in the clinical and epidemiological patterns of number of spores for an individual was estimated. Nosemosis, suggesting that N. ceranae has been one of the 5 PCR analyses of Nosema-positive samples were conduct- most prevalent pathogens in honeybees around the world . ed in the preliminary study before this study11, in which The prevalence of N. ceranae in Iran’s Azerbaijan climatic the samples were taken at the same time. A multiplex-PCR zones was significantly different, and the highest majority method and 16S rRNA genes have been used11. was found in the semi-humid climate (71.00%), followed by very humid (68.10%) and humid (53.80%) climates14. Results and Discussion The number of Nosema spores per bee and climatic condi- As a result of PCR analysis on samples in our previous tions seems to be related to the clinical signs and mortality. study, all species have been identified as Nosema ceranae11. Obvious clinical signs have been higher during the rainy 15 It was found that it had a high-level prevalence in northern months, which we also observed . As a result of the labo- (12.5-62.5%) than in southern in Azerbaijan (0-37.5%). ratory studies, it was defined that the prevalence level of the The study examined 64 samples from eight hives of eight disease is higher in the districts of the region in which the Table 1. Prevalence of N. ceranae according to regions in Azer- humidity is relatively high. Based on these results, it should baijan be noted that there is a proportion between the moisture and the Nosema disease. However, not only the humidity factor but also the other influential factors should be indi- cated as the causes of the disease. These possible factors in- clude the lack of proper care for the bees, dirty water feed- ing, excessive treatment with a high dose of Varroa mite, and other environmental factors. It appears that N. ceranae is better adapted to complete its endogenous cycle with a higher biotic index at different temperatures reflecting the epidemiological differences between both microsporidian species in field conditions and at the colony level16. sites, finding 20 (31.25%) hives Nosema-positive in total. According to the data in the Table 1, the highest infection The highest indicators in the northern region were men- rate of Nosema was detected in Quba and Qusar locali- tioned in Quba and Qusar regions. During the collection ties (62.5%), the least in Bilasuvar and Shabran localities of material from those regions, pesticides have been used (12.5%). No infection was seen in the Masalli area. If re- in the surrounding areas, especially in the apple and cher- viewed by data, the infection in the northern region of the ry gardens. In the Southern part, the highest indicators of country (45.8% average) appears to be greater than in the the disease belong to the Astara region. The high humidity south (22.5% average). level in that region possibly leads to making available con- ditions for creating this disease. 68 Girişgin et al. 2022 gie, 2007; 38(6): 558-565. In previous years, during the studies of the national schol- 9. Huseynov HT. Bio-ecological characteristics of No- ars, the existence of N. apis species has been detected9. sema parasite (Nosema apis Z.) in honey bees of Na- Tahirov and Hüseynov17 reported that the Nosemosis is a khchivan AR, curative and prophylactic actions for it: widespread disease, with the prevalence of N. apis at 24.9- biology candidate of sciences. Dissertation synopsis 48.3% in low coastal areas, 29.8-63.2% in a mountainous of thesis: 03.00.19; The Zoology Institute of ANAS - region and 58.2-87.0% in Zengezur mountainous region of Baku, Azerbaijan. 2006. - 20 p. Nakhichevan Autonomous Republic, only via microscopic 10. Huseynov H, Rustamli Y. In the condition of Nakh- examination. Although there was no molecular diagnosis chivan Autonomous Republic the dependence of in- in those studies, it was possible to see such species of this fection rate of honey bees with Nosema parasite on the disease in many regions of Azerbaijan. The presence of N. strength of colony and the season of the year. III. Na- ceranae in the literal materials was noted in previous stud- khchivan International Beekeeping Conference, 2017, ies in the northwest part of Iran (close to the Azerbaijan Congress book p. 11-15. border) as 48.2%18. In our survey conducted in 2019, the 11. Utuk AE, Aliyeva R, Girisgin AO, et al. First molecu- proliferation of N. ceranae in the southern part of Azerbai- lar detection of Nosema ceranae in Azerbaijan. J Apic jan was described at the molecular level11. Res. 2019; 58(4): 559-561. 12. Shimunaki H, Knox DA. Laboratory diagnosis of hon- This study showed that N. ceranae is a severe and wide- ey bee diseases. US Department of Agriculture, Tech- spread problem in Azerbaijani honeybees. Nosema apis has nical Bulletin, 1990. not been detected in the samples taken from all eight prov- 13. Gurgulova K, Valchovski R, Petrov P, et al. Distribu- inces. Whether in the north or south of the country, the tion of Nosema apis and Nosema ceranae in Bulgar- disease is common in the apiaries and necessary measures ia. Diagnostic in honey bees. From sampling to data should be taken about the disease nationwide. analyses. Beedoc-Cost Action. 2010; Ghent University, Belgium. References 14. Mohammadian B, Bokaie S, Moharrami M, et al. Dis- 1. Anonymous. Azerbaijan Food Balances-2019. The tribution of Nosema spp. in climatic regions of Iran. Food and Agriculture Organization (FAO) Reports, Vet Res Forum. 2018; 9(3): 259 – 263. FAOSTAT. https://www.fao.org/faostat/en/#data/FBS. 15. Aydın L, Gülegen E, Cakmak I, et al. Relation between Accessed April 21, 2022. nosema and chalkbrood diseases, and its implication 2. Higes M, Martin-Hernandez R, Garrido-Bailon E, et for an apiary management model. Bull Vet Inst Pu- al. Detection of infective Nosema ceranae (Microspo- lawy. 2006; 50: 471-475. ridia) spores in corbicular pollen of forager honeybees. 16. Martín-Hernández R, Meana A, Prieto L, et al. Out- J Inverteb Pathol 2008; 97: 76-78. come of colonization of Apis mellifera by Nosema cer- 3. Alaux C, Folschweiller, McDonnell M, et al. Patho- anae. Appl Environ Microbiol. 2007; 73: 6331–6338. logical effects of the microsporidium Nosema ceranae 17. Tahirov A, Huseynov H. Nahcivan Özerk Cumhuri- on honey bee queen physiology (Apis mellifera). J In- yetinde erken ilkbaharda arı kolonilerinin Nosema verteb Pathol. 2011; 106: 380-385. apis ile enfeksiyon derecesine göre gelişme durumları. 4. Forsgren E, Fries I. Comparative virulence of Nosema In: Proceedings of 17th National Parasitology Con- ceranae and Nosema apis in individual European hon- gress and Caucasian and Middle East Symposium on ey bees. Vet Parasitol. 2010; 170: 212-217. Parasitic Diseases, Kars, Turkey, 4–10 September 2011, 5. Fries I. Nosema ceranae in European honeybees (Apis p. 319. mellifera). J Inverteb Pathol. 2010; 103: 73–79. 18. Razmaraii N, Sadigh-Eteghad S, Babaei H, et al. Mo- 6. Klee J, Besana AM, Genersch E, et al. Widespread lecular identification of Nosema species in East Azer- dispersal of the microsporidian Nosema ceranae, an baijan province, Iran. Arch Razi Inst. 2013; 68(1): 23– emergent pathogen of the western honey bee, Apis 27. mellifera. J Inverteb Pathol. 2007; 96: 1–10. 7. Neumann P, Carreck NL. Honey bee colony losses. J Apic Res. 2010; 49(1): 1-6. 8. Paxton RJ, Klee J, Korpela S, et al. Nosema ceranae has infected Apis mellifera in Europe since at least 1998 and may be more virulent than Nosema apis. Apidolo- 69