Detection of Nosema spp. in worker bees, pollen and bee bread during the honey flow season

Nosema apis and Nosema ceranae are responsible for nosemosis in the honey bee (Apis mellifera). The aim of the study was to identify Nosema spp. during the honey flow season in bee colonies, for co-infection or no infection with Nosema apis/Nosema ceranae. Hive bees, forager bees, pollen grains brought by them, and bee bread were analysed. In the infected group, 12 of 30 samples of hive bees were infected with Nosema ceranae, 7 were co-infected, 3 were infected with Nosema apis. In samples of forager bees, 21 of 30 were co-infected, 8 were infected with Nosema ceranae. The analysis of pollen demonstrated that only 2 of 30 samples were infected with Nosema ceranae, 3 of 30 samples of bee bread were infected with Nosema ceranae, 3 were co-infected and 1 of 30 were infected with Nosema apis. In the uninfected group, 13 of 30 samples of hive bees were co-infected, 2 were infected with Nosema ceranae. In the forager bees group, 12 of 30 samples were co-infected, 2 were infected with Nosema ceranae. Only 1 pollen sample was infected with Nosema ceranae and 1 was co-infected, 2 of 30 samples of bee bread were co-infected, 2 were infected with Nosema ceranae. The study demonstrated that during the honey flow season, infection by microsporidian species occurs mainly in forager bees. Pollen and bee bread may be a source of spores mainly at the beginning of the honey flow season. Apiology, coinfections, PCR Nosema apis (N. apis) and Nosema ceranae (N. ceranae) are highly-specialised, parasitic unicellular eukaryotes from the division of Microsporidia (Adl et al. 2005). In the honey bee (Apis mellifera) they are responsible for nosemosis, considered one of the most common adult honey bee diseases in the world. Currently, most honey bees in Europe are infected with N. ceranae, which has also been reported from other regions of the world (Higes et al. 2006; Williams et al. 2008; Michalczyk et al. 2011; MartinHernandez et al. 2012). Bees become infected with the parasite microspores by ingesting water or pollen (Webster et al. 2004; Chen et al. 2008). Weather conditions during the honey flow season play an important role in the development of nosemosis. For N. apis, seasonality of the disease has been observed, while the prevalence of infections with N. ceranae remains on a similar level during the whole honey flow season (Higes et al. 2006, 2007; Paxton et al. 2007; Chen et al. 2009; Bourgeois et al. 2010; Forsgren and Fries 2010). The literature provides very limited data on the sources of bee infection with Nosema spp. spores or the routes of transmission. Pollen and bee bread have been indicated as potential sources of infection (Higes et al. 2008a). Disease in the apiary and bee colony is usually transmitted via combs infected with spores, water, and between insects by means of trophallaxis (OIE 2013). The aim of our study was to identify Nosema spp. present during the honey flow season in infected (co-infected with N. apis/N. ceranae) and uninfected honey bee colonies. The analysis included 2 castes of worker bees: bees working on combs sampled directly from the central part of the nest, and foragers bringing pollen to the hive, as well as pollen grains collected by foragers and bee bread stored in newly built combs. ACTA VET. BRNO 2016, 85: 261-266; doi:10.2754/avb201685030261 Address for correspondence: Maria Michalczyk Departament of Parasitology and Invasive Diseases Faculty of Veterinary Medicine, University of Warmia and Mazury Oczapowskiego 13 Street, 10-718 Olsztyn, Poland E-mail: maria.michalczyk@uwm.edu. pl Phone: +48 89 523 35 77 http://actavet.vfu. cz/ Materials and Methods Material for the study was sampled in March 2014 from a 50-hive apiary (53°47’N, 20°30’E). A haemocytometric method (Neubauer chamber) was used to detect Nosema spp. spores in each honey bee colony by analysing 60 worker bees randomly sampled from winter hive debris. Multiplex polymerase chain reaction (PCR) demonstrated that 90% of positive samples were co-infected with N. apis/N. ceranae. Therefore, further tests were carried out on 5 honey bee colonies (group 1) without nosemosis symptoms, infected with Nosema spp. (more than 1.2 × 106 spores/worker bee), and with a co-infection confirmed by PCR (N. apis/N. ceranae) and 5 bee colonies (group 2 – control) free from microsporidians. Every month from April to September 60 hive bees (HB) were sampled randomly directly from honey combs in the central part of the nest, and 60 forager bees returning to the hive with pollen (FB) were sampled from the beehive entrance using a special tool. Multiplex PCR was used to separately analyse worker bees and pollen brought by them (Sokół and Michalczyk 2012). Pollen grains from forager bees were collected using sterile tweezers. A single sample contained 20 randomly collected pollen grains. In addition, about 5 g samples of bee bread stored in newly built marked combs were collected. Fifteen mg samples of bee bread were used for analysis. Deoxyribonucleic acid (DNA) was isolated from bees, pollen and bee bread using the same technique. Genomic DNA was isolated using the Genomic Mini A&A Biotechnology kit (Gdynia, Poland) for DNA extraction by relying on genomic DNA’s ability to bind to silica in the presence of high concentrations of chaotropic salts. The isolation process was carried out in accordance with the manufacturer’s guidelines, and purified DNA was stored for further analyses in test tubes at a temperature of -20 °C. The multiplex PCR assay involved the amplification of small subunit ribosomal ribonucleic acid (rRNA) sequences (16S rRNA) of Nosema apis and Nosema ceranae. The following primers were used: for N. ceranae MITOC FOR (5’-CGGCGACGATGTGATATGAAAATATTAA-3’) and MITOC REV (5’-CCCGGTCATTCTCAAACAAAAAACCG-3’) amplifying a 218 bp product for N. apis and 321 bp for N. ceranae (OIE 2013). Multiplex PCR analysis was carried out using the HotStarTaq Plus Polymerase (Qiagen, Germany) and the HotStarTaq Plus Master Mix Kit (Qiagen, Germany). The reaction mix of 20 μl comprised around 120 ng isolated DNA, 10 μl HotStarTaq Plus Master Mix 2 times, 2 μl CoralLoad Concentrate × 10 and 0.1 μl of each primer, supplemented with RNase-Free Water to 20 μl. Every reaction involved three controls: two positive controls with the DNA of Nosema apis and Nosema ceranae (Centro Nosema Apicola Regional, Dirección Genera de la Producción Agropecuaria, Consejería de Agricultura, Junta de Comunidades de Castilla-La Mancha, Spain) and one negative control where DNA was replaced with water. The reaction was carried out in the Eppendorf Mastercycler thermocycler. The PCR commenced with initial denaturation at 95 °C for 5 min. The reaction mixture was then cycled × 35 consisting of the following steps: denaturation at 94 °C for 45 s, primer annealing at 55 °C for 45 s and extension at 72 °C for 1 min. The last reaction was followed by final chain synthesis at 72 °C for 10 min. The products of the multiplex PCR reaction were separated by electrophoresis in 2% agarose gel containing 0.5 μg/ml ethidium bromide for visualizing the resulting DNA fragments of Nosema spp. The size of the obtained products was evaluated by comparison with the GeneRulerTM 100 bp 36 Ladder Plus (Fermentas, USA) molecular size marker. Electrophoresis results were archived using the GelDoc (Bio-Rad, USA) gel documentation system.