The growing prevalence of Nosema ceranae in honey bees in Spain, an emerging problem for the last decade Cristina Botías a , Raquel Martín-Hernández a,b , Encarna Garrido-Bailón a , Amelia González-Porto a , Amparo Martínez-Salvador c , Pilar De La Rúa d , Aránzazu Meana e , Mariano Higes a,⇑ a Centro Apícola Regional (CAR), Dirección General de la Producción Agropecuaria, Consejería de Agricultura, Junta de Comunidades de Castilla-La Mancha, 19180 Marchamalo, Spain b Instituto de Recursos Humanos Para la Ciencia y la Tecnología (INCRECYT), Fundación Parque Científico y Tecnológico de Albacete (ESF), Spain c Departamento de Epidemiología, TRAGSATEC, Madrid, Spain d Departamento de Zoología y Antropología Física, Facultad de Veterinaria, Universidad de Murcia, 30071 Murcia, Spain e Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain article info Article history: Received 30 May 2011 Accepted 9 August 2011 Keywords: Nosema ceranae Nosema apis Honey Nosemosis type C Epidemic status abstract Microsporidiosis caused by infection with Nosema apis or Nosema ceranae has become one of the most widespread diseases of honey bees and can cause important economic losses for beekeepers. Honey can be contaminated by spores of both species and it has been reported as a suitable matrix to study the field prevalence of other honey bee sporulated pathogens. Historical honey sample collections from the CAR laboratory (Centro Apícola Regional) were analyzed by PCR to identify the earliest instance of emergence, and to determine whether the presence of Nosema spp. in honey was linked to the spread of these microsporidia in honey bee apiaries. A total of 240 frozen honey samples were analyzed by PCR and the results compared with rates of Nosema spp. infection in worker bee samples from different years and geographical areas. The presence of Nosema spp. in hive-stored honey from naturally infected honey bee colonies (from an experimental apiary) was also monitored, and although collected honey bees resulted in a more suitable sample to study the presence of microsporidian parasites in the colonies, a high probability of finding Nosema spp. in their hive-stored honey was observed. The first honey sample in which N. ceranae was detected dates back to the year 2000. In subsequent years, the number of sam- ples containing N. ceranae tended to increase, as did the detection of Nosema spp. in adult worker bees. The presence of N. ceranae as early as 2000, long before generalized bee depopulation and colony losses in 2004 may be consistent with a long incubation period for nosemosis type C or related with other unknown factors. The current prevalence of nosemosis, primarily due to N. ceranae, has reached epidemic levels in Spain as confirmed by the analysis of worker honey bees and commercial honey. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Microsporidiosis caused by microsporidia of the genus Nosema is one of the most widespread disease of adult honey bees, and it is responsible for significant economic losses for beekeepers world- wide acting alone (Higes et al., 2008a, 2009; Hornitzky, 2008; Heintz et al., 2011) or in combination with other synergistic factors (Alaux et al., 2010; Bromenshenk et al., 2010; Bacandritsos et al., 2010; Vidau et al., 2011). Two species of microsporidia infect Apis mellifera, namely Nosema apis and Nosema ceranae. N. apis was first described over a 100 years ago, and its effects on individual bees and whole colonies have been widely documented (revised by Neveu-Lemaire (1938); Bailey and Ball (1991); OIE (2008)). By contrast, N. ceranae is a microsporidium of the Asian bee Apis cerana (Fries et al., 1996) that was recently shown to parasitize A. mellifera (Higes et al., 2006; Huang et al., 2007) and is now detected worldwide as one of the pre- dominant microsporidia infecting honey bees (Martín-Hernández et al., 2007; Chaimanee et al., 2010; Chen and Huang, 2010; Higes et al., 2010a; Suwannapong et al., 2010; Tlak Gajger et al., 2010; Stevanovic et al., 2011), even though it seems that N. apis may be more prevalent in colder climates (reviewed by Fries (2010); Gisder et al. (2010a)). Both microsporidia are obligate intracellular para- sites that infect epithelial cells of the honey bee ventriculum after ingestion of spores, although they cause two distinct illnesses with different epidemiological patterns, clinical signs, and consequences in terms of honey bee and colony viability (Chen and Huang, 2010; Gisder et al., 2010b; reviewed in Higes et al., 2010a; Bourgeois et al., 2011). Indeed, there are data suggesting that N. ceranae is more adaptable and less host specific than N. apis (Plischuk et al., 2009; Martín-Hernández et al., 2009; Chaimanee et al., 2010; Suwanna- pong et al., 2010; Chen and Huang, 2010; Higes et al., 2010b). Hence, it has been recently proposed to distinguish between the nosemosis caused by N. apis (nosemosis type A) and the disease caused by 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.08.002 ⇑ Corresponding author. Tel.: +34 949 250026; fax: +34 949 250176. E-mail address: mhiges@jccm.es (M. Higes). Research in Veterinary Science 93 (2012) 150–155 Contents lists available at SciVerse ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc