Distribution of Nosema Spp. in climatic regions of Iran.

‎Nosemosis is one of the most prevalent bee diseases in the world causing significant ‎economic losses in the global bee-keeping industry. This cross-sectional study was conducted during April-September, 2016 to investigate the prevalence of nosemosis in different climatic regions of Iran. A total of 183 ‎apiaries were selected based on cluster sampling and the climate of apiaries under study was classified using Domarten method. In each apiary, five percent of the colonies were ‎ randomly sampled. A total of 183 adult bee samples were taken and examined by microscopic and polymerase chain reaction (PCR) methods for the presence of Nosema infections. According to the results, infection caused by Nosema ceranae was observed in all regions under study. The prevalence of N. ceranae was 46.40% (42.70-50.10). However, infection with Nosema apis was not observed in the samples in either pure form or as ‎associated infection. Based on the results of PCR, the prevalence of N. ceranae was ‎53.80% (46.60- 61.00) ‎ in ‎humid, ‎71.00% (53.70-77.50) ‎ in semi-humid‎, ‎68.10% (61.40-74.80) ‎ in very humid, ‎29.40% (22.70-36.10) in arid, ‎34.30% (27.40-41.20) in semi-‎arid and ‎24.00% (17.90-30.00)‎ in Mediterranean climates. The prevalence of ‎infection in different climatic zones of the country was found to have significant ‎differences (p < 0.001). ‎ According to the findings, N. ceranae ‎ was the only Nosema species in honeybees with a broad geographical dispersion in Iran. It seems that climate can influence the prevalence of mentioned parasite. ‎.

Introduction

Nosemosis is known as an important bee disease with worldwide distribution. It is an infectious disease of honeybees that leads to the weakening and ‎eventually death of colony.[1] The disease was first described by Zander‎‎ (1909), although the spores had been shown to cause the disease in much earlier.[2] ‎Nosema spores are not relatively sensitive to heat and drought and survive in unfavourable ‎environmental conditions for a long time.[3] The disease is ‎transferred horizontally by worker bees during ingestion of the spores when cleaning the ‎fecal stains on the frames.[4]

Two species known as N. ceranae and N. apis are pathogenic for bees. All the bees in a colony‎‎ (queen, workers and male bees) are vulnerable to be infected with Nosema species.[5] The spores of these species share similar morphological characteristic making the distinction quite difficult by traditional microscopic methods.[6] Recently, the polymerase chain reaction (PCR) technique has been used for successful detection of the entire life cycle of the microsporidia and determines the parasite even at a very ‎low level of microsporidia infection.[7]

Since Nosema apis has been the pathogen of European honey bees for several years, numerous studies have been conducted on nosemosis with N. apis agent, but studies on N. ceranae are limited.[8] The studies carried out before 2003 suggest the worldwide expansion of N. apis in A. mellifera, though N. ‎ceranae has been shown to spread among A. mellifera in Europe, America and Asia after 2003.[9]

Higes et al. for the first time have reported the natural infection of A. melifera with N. ceranae among the apiaries in Spain.[10] However, it was found that N. ceranae is not a new pathogen for European honeybee and its previously unknown nature may have been due to the difficulty of differential diagnosis of the Nosema species.[11]

N. ceranae was first reported by Nabian et al. among A. mellifera colonies in Mazandaran, ‎Iran.[12] later, the infection with the parasite has been reported by further studies. The ‎study by Razmaraii et al. showed that N. ceranae was the only Nosema species in ‎East Azerbaijan province.[13] According to the findings of a study by Modirrousta et al., the ‎samples collected from five provinces in Iran from 2004 to 2013 were positive for N. ceranae. The ‎identification of N. ceranae in the samples of recent years in Iran indicates the transition of N. ceranae ‎ to A. mellifera before its identification.[14] Besides, the study by Aroee et al. ‎reported the infection of honeybee samples with N. ceranae microsporidia in three provinces of Esfahan, Chaharmahal and ‎Bakhtiari, and Fars.[15]

Since N. ceranae infection has been reported in different regions of Iran in the recent years, this study was conducted to estimate the dispersion of Nosema Spp. in different climatic regions of the Iran.

Materials and Methods

This cross-sectional study was performed during April-September, 2016. A total of 183 ‎apiaries located in different provinces of Iran were selected by cluster sampling (Table 1). The climate of selected apiaries was determined based on Domarten approach. In this approach, the climate of region is assessed based on the following drought indicator: [16]

Table 1

Frequency of studied apiaries in different provinces of Iran in 2016.

Provinces	No. of apiaries *	Frequency	Valid (%)
Kerman	194	2	1
Qom	187	2	1
Yazd	668	9	5
South Khorasan	337	2	1
Khorasan Razavi	1078	8	4
Lorestan	1808	13	7
North Khorasan	1400	4	2
Chaharmahal and ‎ Bakhtiari 1268		7	4
Fars	2688	14	8
West Azerbaijan	3882	26	14
Kurdistan	3606	22	12
Gilan	2579	17	9
Ardabil	705	4	3
Qazvin	1344	10	5
Golestan	1824	13	7
Mazandaran	3160	21	12
Semnan	283	9	5
Total	27011	183	100

Iran Veterinary Organization data (2015).

Aridity Index = P/T + 10

where, P is the average annual rainfall (mm) and T is normal rate of annual temperature (˚C). In this study, the type of climates was determined by calculating the drought indicator of each region.

Sample collection. In each apiary, five percent of colonies were ‎sampled randomly. All samples were stored at –20 ˚C before analyzed. ‎Adult bee samples were then examined for the presence of Nosema infections using ‎molecular and microscopic ‎diagnostic approaches.

Preparation of samples for microscopic examination. The abdomen of 20 adult dead honey‎bees from each apiary were macerated in 10 mL distilled water and crushed in a mortar. Then, the ‎suspension was passed through a 100 µm mesh sieve to remove the debris and ‎centrifuged for 6 min at 800 g. Finally, the supernatant was discarded and the pellet was ‎examined under a common light microscope at 400× magnification.[17]

Preparation of samples for PCR. The abdomen of 20 adult dead honeybees from each apiary ‎were macerated in 10 mL distilled water (PCR grade) and the suspension was then filtered and ‎centrifuged at 800 g for 6 min. Spore germination was induced with 200 μL freshly prepared ‎germination buffer and the mixture was incubated at 37 ˚C for 15 min.17

DNA extraction. The DNA was isolated using DNA extraction kit (Takapozist, Tehran, Iran) according to the ‎manufacturer’s instructions.

PCR. Amplification was performed by the PCR kit (Sinacolon, Tehran, Iran) in a Mastercycler gradient (Eppendorf, Hamburg, Germany) under conditions as follows: 50 μL reaction cocktail containing 25 μL of high-fidelity PCR master mixture, 0.40 μM of each primer, 0.40 mM of each deoxynucleoside triphosphate, 3 mM Cl2Mg, 0.20 mg mL-1 bovine serum albumin, 0.10 Triton X-100 and 5 μL N. apis or N. ceranae DNA template, initial denaturation at 94 ˚C for 2 min, 10 cycles of 94 ˚C for 15 sec, 61.80 ˚C for 30 sec and 72 ˚C for 45 sec and 20 cycles of 94 ˚C for 15 sec, 61.80 ˚C for 30 sec and 72 ˚C for 50 sec along with the final extension at 72 ˚C for 50 sec and at 72 ˚C for 7 min. Amplicons were run on a 2.00% agarose gel electrophoresis, stained with a safe stain and visualized by ultraviolet transillumination.7 In this study, a small subunit 16S rRNA gene of N. apis and N. ceranae was used (Table 2). Positive controls for the mentioned species were prepared from Honeybee-Silkworm and Wildlife Diseases’ Department of ‎ Razi Vaccine and Serum Research Institute, Karaj, Iran.‎ The results of PCR are shown in Fig. 1.

Table 2

The sequence of specific primers used for identification of Nosema species in PCR according to Martín-Hernández et al.7

Specificity	Primer sequence (5`-3`)	PCR product size (bp)
N. ceranae	FWD: 5'- CGGCGCGACGATGTGATATGAAAATATTAA-3'	218-219
	REV: 5'-CCCGGTCATTCTCAAAAAACCG-3'
N. apis	FWD: 5'-GGGGGCATGTCTTTGACGTACTATGTA-3'	321
	REV: 5'-GGGGGCGTTTAAAATGTGAAACAACTATG-3'

Fig. 1

The PCR results of N. ceranae detection. 1: Lane Marker, 2: Negative control, 3: Positive control (N. apis & N. ceranae), 4, 5, 6, 7 (N. ceranae).

Data Analysis. Descriptive results were prepared using SPSS (version 21.0; SPSS Inc., Chicago, USA). The chi-square test was used to compare the level of infection with N. ceranae in all regions under study. Cohen’s kappa value was used to evaluate the agreement between molecular and microscopic tests by SPSS.

Results

Based on the findings, N. apis was not found in any of the samples. In contrast, N.‎ ceranae parasite was found in all studied regions.

The prevalence of N. ceranae was 46.40%. Table 3 shows the prevalence of N. ceranae based on microscopic and molecular diagnostic approaches. The confidence interval of total prevalence was calculated based on the 183 samples in the climatic regions‎ of the country. The results indicated that contamination with N. ceranae was‎ significantly different in various climatic regions of the country (p < 0.001).

Table 3

The prevalence of Nosema ceranea in climatic regions of Iran in 2016.

Climate	No. of Sample	Molecular results		Microscopic results
		No. of positive	Prevalence (CI: 95%)	No. of positive	Prevalence (CI: 95%)
Total	183	85	46.40% (42.70–50.10)	66	36.10% (29.20–43.00)
Humid	39	21	53.80% (46.60–61.00)	15	38.50% (31.50–45.50)
Semi-humid	31	22	71.00% (53.70–77.50)	21	67.70% (60.90–74.50)
Very Humid	22	15	68.10% (61.40–74.80)	14	63.60% (56.60–70.50)
Mediterranean	25	6	24.00% (17.90–30.00)	2	8.00% (4.00–11.90)
Arid	34	10	29.40% (22.70–36.10)	6	17.60% (12.10–23.10)
Semi-arid	32	11	34.30% (27.40–41.20)	8	25.00% (18.70–31.30)

The Kappa coefficient ‎was also calculated in order to determine agreement between microscopic and molecular tests. The kappa value was 0.79 (0.65–0.93), thus the concordance between the two methods was substantial.

Discussion

Nowadays, infection with N. ceranae has been proved in all continents. Recent reports have confirmed changes in the clinical and ‎epidemiological patterns of nosemosis suggesting that N. ceranae has been ‎one of the most prevalent pathogens in honeybees around the world,[18] which is extremely virulent for its new host, A. mellifera.[5]

Earlier reports in Iran have indicated that N. ‎apis has been the only pathogen of nosemosis.[12] Studies conducted in Iran ‎reported N. ‎apis infection in honeybee colonies in Arasbaran,[19] East ‎Azerbaijan province,20 Tabriz and Meshkinshahr,[21] ‎Urmia[22] and North Khorasan province.[23]

In contrast to previous studies, the findings of the present study indicated a widespread prevalence of N. ceranae in Iran. The ‎experimental results indicated pure infection with N. ceranae in 85‎‎ (46.40%) apiaries. The absence of N. apis in its main host, A. mellifera, could mean the substitution of N. ‎ceranae in Iranian apiaries. Although it is not recognized when and how the host shift‎ happened, several reasons have been proposed including the transfer of ‎honeybees and queen products between apiaries, migration and climate change in the recent years.

According to a study in Turkey, Van province, the prevalence of nosemosis was identified to be 32.50%. However, N. ceranae was the only Nosema species found to infect honeybees and no N. apis or mixed infections were detected in the samples.[24]

Similarly, different studies ‎conducted in Croatia,[25] Taiwan,[26] Uruguay[27] and the United States[7],[28] confirmed the transition of N. ‎ceranae to A. mellifera. In contrast, ‎there have been no reports of substitution of parasite with another one in countries such as Germany ‎and Sweden.[18]

In this study, the prevalence of N. ceranae in the climatic zones ‎was significantly different and the highest prevalence was found for the semi-humid climate (71.00%) followed by very humid (68.10%) and humid (53.80%) climates. ‎The high prevalence of infection in the humid regions is in accordance with the ‎findings of the study conducted in Turkey indicating that the impact of moisture on N. ceranae ‎is more intense in areas with higher humidity and the humidity level was described ‎as a vital factor for ‎Nosema species.[29] This finding may allow us to predict the dynamics of N. ceranae using average humidity in ‎different regions. ‎

According to De la Rocque et al., climatic changes may influence ‎distribution, intensity and seasonality of infectious diseases such as nosemosis.[30] The multiplication rates of both ‎parasites (N. ceranae and N. apis) are dependent on temperature but N. ceranae exhibits a faster growth in slightly higher ‎temperatures compared to N. apis.[31] Higher ‎prevalence of N. ceranae in warmer weather was proposed by Fries and Forsgren, while the study by Higes et al. reported the low seasonal dependence of N. ceranae ‎and its presence in all four seasons.[8]

The study by Martín-Hernández et al. also emphasized ‎the presence of the parasite throughout the year,[7] while a study by Stevanovic et al. ‎showed a seasonal pattern of the aforesaid parasite.[32]

Nosema ceranae infection has been found to have a wide range of clinical symptoms. Also, since several ‎cases of colony population decline have been attributed to the infection of bees with N. ceranae,[33]-[36] it is necessary to study the causal relation of N. ceranae ‎with the sudden decline in the bee population in apiaries across the country.

According to the findings of present study, N. ceranae was the main causal agent of nosemosis in the studied apiaries of Iran. It seems that N. ceranae has been ‎transferred to its new host, A. mellifera. Since the prevalence of the parasite was significantly different among climatic regions, this probably enables to predict the dynamics of N. ceranae using climatic factors in ‎different regions.‎