Journal of Invertebrate Pathology 111 (2012) 106–110

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Journal of Invertebrate Pathology

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Asymptomatic presence of Nosema spp. in Spanish commercial apiaries

José Manuel Fernández a,⇑, Francisco Puerta b, Mercedes Cousinou c, Rafaela Dios-Palomares d,Francisco Campano b, Laura Redondo c

a Apicultural Reference Center in Andalusia (C.E.R.A.), Spainb Andalusian Beekeeping Center, Cordova University, Spainc Department of Genomics, Central Service of Support for Research, Cordova University, Spaind Department of Statistics, Efficiency and Productivity Group EFIUCO, Cordova University, Spain

a r t i c l e i n f o

Article history:Received 9 November 2011Accepted 26 June 2012Available online 20 July 2012

Keywords:Nosema apisNosema ceranaeHoney beesCCDPCRCapillary electrophoresis

0022-2011/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.jip.2012.06.008

⇑ Corresponding author. Address: Campus UniversSan José 7, 14071 Córdoba, Spain.

E-mail addresses: zo3fepej@gmail.com, zo3fepej@uURL: http://www.centroapicola.com (J.M. Fernánd

a b s t r a c t

Nosemosis is caused by intracellular parasites (Nosema apis and Nosema ceranae) that infect the midgutepithelial cells in adult honey bees. Recent studies relate N. ceranae to Colony Collapse Disorder and thereis some suggestion that Nosema spp., especially N. ceranae, induces high mortality in honey bees, a factthat is considered as a serious threat for colony survival.

604 samples of adult honey bees for Nosema spp. analysis were collected from beekeeping coloniesacross Spain and were analysed using PCR with capillary electrophoresis. We also monitored 77 Andalu-sian apiaries for 2 years; the sampled hives were standard healthy colonies, without any special diseasesymptoms.

We found 100% presence of Nosema spp. in some locations, indicating that this parasite was widespreadthroughout the country. The two year monitoring indicated that 87% of the hives with Nosema spp.remained viable, with normal honey production and biological development during this period of time.The results of these trials indicated that both N. ceranae and N. apis could be present in these beehiveswithout causing disease symptom and that there is no evidence for the replacement of N. apis by N. cer-anae, supporting the hypothesis that nosemosis is not the main reason of the collapse and death ofbeehives.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Nosemosis is a parasitic disease that attacks all adult forms ofhoney bees: workers, queens and drones (Bailey, 1955; Alauxet al., 2011; Traver and Fell, 2011). The disease is caused by themicrosporidia Nosema apis and/or Nosema ceranae (Bailey, 1962;Fries et al., 1996). As a mechanism of infection, microsporidia aremainly characterised by their ability to disperse among their hostas spores. Inside the host the spores germinate and the sporoplasmenter the host cell via the polar filament which is injected into thehost cell (Kudo, 1954; Fries et al., 1996, 2006; Lee et al., 2008).There are other microsporidia species that can parasites insects(Avery and Anthony, 1983; Bomar et al., 1993), reptiles (Jacobsonet al. 1998), birds (Dorrestein and van der Hage, 1999), fishes(Lom and Nilsen, 2003) and mammals (Percy and Barthold, 1993;Wasson and Peper, 2000), including humans (Shadduck et al.,1990; Cali, 1991; Didier, 2005).

ll rights reserved.

itario de Rabanales, Colonia

co.es (J.M. Fernández).ez).

Both N. apis and N. ceranae develop inside the epithelial cells ofthe bee’s ventriculus, interfering with the absorption of proteins(Liu, 1984; Puerta et al., 2001; Chen et al., 2009). Although this par-asite is essentially distributed worldwide (Matheson, 1996; Chenet al., 2007; Higes et al., 2007; Klee et al., 2007; Liu et al., 2008;Williams et al., 2008), the virulence of both species is controversial(Paxton, 2010), and it was also suggested that N. ceranae couldcause colony collapse (Cox-Foster et al., 2007; Higes et al., 2008,2009b).

Over the last decade, Colony Collapse Disorder (CCD) is hav-ing a massive impact in Spain, with approximately 30% of theSpanish hives being lost in recent years (unofficial data frombeekeepers’ associations). Spain has the largest number of bee-hives (2,300,000 hives) in the European Union, and Andalusiais the Spanish region that has the largest number of hives(442,466 hives). Due to this large amount of beehives, CCD hasbecome more important in this country in which the incomeof 80% of professional beekeepers (having more than 150 hives)depends heavily on beekeeping (General Register of LivestockFarms. Ministry of the Environment, Rural and Marine Affairs,2008).

J.M. Fernández et al. / Journal of Invertebrate Pathology 111 (2012) 106–110 107

In response to the increasing concern over CCD, we moni-tored samples of honey bee hives in the main Spanish areaswhere beekeeping is practised professionally to clarify the roleof nosemosis in CCD and to determinate whether N. ceranaecauses this disorder.

2. Materials and methods

2.1. Sampling

Samples were collected and monitored by the beekeeping tech-nicians of the different livestock organisations and health advisoryassociations. Each sample consisted of 100 adult bees collectedfrom the outer honey frame of the same hive. The samples werestored in a freezer at �20 �C until analysis.

The hives chosen did not show any clinical symptoms and didnot receive any antifungal treatment, but they were treated withauthorised Varroa destructor products at least once a year. Thehives were randomly selected and identified for further samplecollection for follow up of the hive’s status over time. In addition,the beekeeper completed a questionnaire.

There were two types of sampling:

(a) Monitoring of 77 Andalusian apiaries (one hive per apiary)during 2006–2007. Samples were collected twice a year inthe spring and autumn. If any of the beehives died duringthe sampling period they were randomly replaced by otherdisease symptom-free hives belonging to the same apiaryfor future studies.

Table 1Detection of Nosema spp. in asymptomatic colonies in Andalusia (Southern Spain) during

Andalusia

Period of sampling Province N� samples N. ceranae N. api

No. samples (%) No. sa

Spring 2006 Almeria 10 5 50 1Cadiz 6 4 66.6 1Cordova 10 7 70 –Granada 10 7 70 –Huelva 8 7 87.5 –Jaen 7 6 85.7 –Malaga 11 9 81.8 –Seville 8 8 100 –

Autumn 2006 Almeria 10 7 70 –Cadiz 8 6 75 –Cordova 9 6 66.6 –Granada 9 6 66.6 –Huelva 5 5 100 –Jaen 6 6 100 –Malaga 1 1 100 –Seville 6 6 100 –

Spring 2007 Almeria 35 15 43 5Cadiz 32 27 85 1Cordova 34 34 100 –Granada 35 19 54 2Huelva 35 34 97 –Jaen 33 33 100 –Malaga 34 24 71 –Seville 33 25 76 –

Autumn 2007 Almeria 9 7 78 1Cadiz 8 5 62 –Cordova 6 6 100 –Granada 3 2 67 –Huelva 8 8 100 –Jaen 9 9 100 –Malaga 5 5 100 –Seville 3 3 100 –

Total andalusia 446 352 78.9 11

(b) To determine the presence of Nosema spp., five different bee-hives from each of 118 apiaries around the country werealso sampled during 2007.

2.2. DNA extraction

Fifteen bee abdomens of each sample were macerated in 10 mlof Milli-Q ultrapure water and filtered through a sieve of 0.036 mmin diameter. The filtered suspension was processed using theDNeasy Plant Mini Kit (Quiagen�).

2.3. DNA amplification

The amplified regions belonged to a gene sequence of the 16Sribosomal RNA subunit: the N. apis region is 242 bp. and N. ceranaeregion is 254 bp. This size difference allowed the identification ofboth species by PCR.

The primers used for the amplification reaction were describedby Higes et al. (2006). Our research team used the same NOS-REVprimer and changed a pair of bases of the NOS-FOR primer;two bases were added to the 50 end of the sequence thatappeared in the database, and the last two bases of the 30 endwere removed because there were differences in these nucleo-tides between the N. apis and N. ceranae (U26534, GI857487;U26533, GI857489) sequences. The primer sequence used was50-TATGCCGACGATGTGATATG-30.

These primers were used to sequence the products obtained byPCR and to confirm the sequences in both species. Sequencing wasperformed with the BigDye Terminator v 3.1 Cycle Sequencing kit

2006 and 2007.

s Coinfection (N. apis + N. ceranae) Not detection

mples (%) No. samples (%) No. samples (%)

10 4 40 – –16.7 1 16.7 – –– 3 30 – –– 3 30 – –– 1 12.5 – –– 1 14.3 – –– 2 18.2 – –– – – – –

– 3 30 – –– 2 25 – –– 3 33.3 – –– 3 33.3 – –– – – – –– – – – –– – – – –– – – – –

14 12 34 3 93 3 9 1 3– – – – -6 13 37 1 3– – – 1 3– – – – –– 9 26 1 3– 7 21 1 3

11 1 11 – –– 1 13 2 25– – – – –– – – 1 33– – – – –– – – – –– – – – –– – – – –

2.5 72 16.1 11 2.5

Nosema spp. in Andalusia

0

20

40

60

80

100

Spring 2006 Autumn 2006 Spring 2007 Autumn 2007

Samplig period

Perc

ent o

f col

onie

s

N. ceranaeN. apisCo-infectionNo detection

Fig. 1. Percent of colonies infected with Nosema spp. in Andalusia during 2006(n = 96) and 2007 (n = 322).

108 J.M. Fernández et al. / Journal of Invertebrate Pathology 111 (2012) 106–110

from Applied Biosystems using an automatic sequencer 3130XLGenetic Analyzer (Applied Biosystems).

A GeneAmp PCR System 9700 (Applied Biosystems) was used forthe amplification reaction. The steps of the reaction were as follows:

- An initial denaturation step at 94 �C for 2 min.- Thirty-five cycles of denaturation at 94 �C during 30 s, hybrid-

isation at 60 �C for 30 s and polymerisation at 72 �C during 30 s.- A final extension step at 72 �C for 4 min.

The primer ‘‘Forward’’ was labelled with the 6-Fam blue fluoro-phore to detect the PCR products using capillary electrophoresiswith a 3130XL Genetic Analyzer and the GeneMapper v3.7. Pro-gram. For the electrophoresis, 2 ll of the obtained products weremixed with deionised formamide Hi-di from Applied Biosystems;size standards of molecular weights between 50 and 400 bp (Gene-Scan Rox 400HD) were included.

A sample from the C.E.R.A. experimental apiary of Cordova Uni-versity was used as a N. ceranae positive control in each PCR reac-tion. Dr. Robert J. Paxton from Queen’s University Belfast supplieda N. apis positive control; water was used as the negative control.

n= 446

n=

n= 24

n= 8

58%

42%

87%

13%

79%

2.5%16%2.5%

56%

8%16%

4%72%

Fig. 2. Distribution of Nosema spp. in Sp

3. Results and discussion

The survey data from our 2 years of beehive monitoring indicatethat all of the samples tested were positive for Nosema spp. (Ta-ble 1), that is, the presence of N. apis, N. ceranae or both. Of themonitored hives, 87% remained alive, with normal production dur-ing the study. According to the beekeeper’s questionnaire, the mainreason for the death of the monitored samples was a V. destructorinfestation.

Both N. apis and N. ceranae were present in hives in springand autumn, sometimes as a coinfection, sometimes as a singleinfection by one species only. Our results are contradictory tothose described by other authors (Martin-Hernandez et al.,2007; Higes et al., 2007, 2008, 2009b), who suggested that N.ceranae causes colony collapse within 18 months of a colonybecoming infected and infections left uncontrolled. The resultsof the Andalusian hives monitored over 2 years showed thatthe same hive parasitised by Nosema spp. remained alive duringthose 2 years without collapsing. The data obtained in the pres-ent study are complementary and consistent with the results re-ported by Invernizzia et al. (2009), whose hives were monitoredfor decades and demostrated no correlation between the arrivalof N. ceranae, the presence of it in the hives or hive losses due toCCD. The same situation was shown in Germany (Siede et al.,2008) as the monitored hives (Monitoring-Projekt ‘‘Völkerverlu-ste’’, 2008) suggested no such devastating effects as describedby Higes et al., 2009a.

The prevalence of N. ceranae appears to be much higher in Spaincompared to N. apis in the present study (Fig. 1), which is congru-ent with earlier findings from Europe, including Spain (Martin-Hernandez et al., 2007). However, our results do not demonstratea continuous replacement of one parasite for the other, as sug-gested by several studies (Paxton et al., 2007; Higes et al., 2007,2008, 2009b). In this study, we found no pattern where the pres-ence of N. ceranae in the spring reduced the probability of findingN. apis in the fall, or vice versa. Clearly, honey bee colonies can be-come infected by either of the two parasites or co-infected by both,

n= 16

n= 20

n= 40

25 n= 25

44%

45%

30% 15%

10%

40%

50%10%

52% 4%

32%12%

N. ceranae

No detection

Coinfection

N. apis

anish professional apiaries. n = 604.

J.M. Fernández et al. / Journal of Invertebrate Pathology 111 (2012) 106–110 109

without necessarily shifting from one parasite to the other (Paxton,2010). An increased incidence of microsporidia infections in recentyears has been reported from Spain (Fig. 2), with this increaseapparently due to increased prevalence of N. ceranae (Higes et al.,2008). However, N. ceranae appears to have no competitive advan-tage in co-infected bees (Forsgren and Fries, 2010) and N. apisinfections are still present in the Spanish honey bee population.And obviously both infections can be present in honey bee colonieswithout causing depopulation or colony collapse.

Fig. 2 shows a map of the Spanish Nosema spp. distribution builtthrough the analysis of 604 samples from most of the Spanishterritory.

Regarding the distribution of microsporidia, its presence isremarkable throughout Spain, as all locations were positive for N.ceranae (see Table 2 in the complementary information section).However, the presence of N. apis nor of N. ceranae in beehives ap-pear to result in colony collapse (Paxton, 2010).

N. ceranae has coexisted with Apis mellifera in Europe for morethan a decade (Klee et al., 2007), at least from the mid-1990s inthe US Chen et al., 2008) and in Uruguay infections with N. ceranaepre-dates 1990 (Invernizzia et al., 2009). A lack of historic samplesmakes it impossible to conclude for how long N. ceranae has parasi-tised A. mellifera, but available data strongly suggest that this par-asite can be present in honey bee colonies for extended periodswithout causing symptoms or colony losses (Paxton, 2010). Micro-sporidia infections are common in honey bees and is often presentin honey bee colonies without causing disease symptoms at thecolony level. It is under adverse conditions that the host-parasiterelationship results in disease outbreak and colony damages (Fries,2010; Puerta et al., 2001).

In the questionnaire completed by beekeepers, 84% of the bee-keepers claimed to have problems with the ectoparasite Varroadestructor. Infestations by this mite, with its associated virus infec-tions, is still the major culprit explaining a substantial proportionof colony losses world-wide (Dahle, 2010; Guzman-Novoa et al.,2010; Le Conte et al., 2010).

Acknowledgments

The authors would like to thank the Functional Genomic Unit(University of Cordova), Spanish Farming Association (COAG) andM.C. Fernández Perejón for their editing, comments and sugges-tions. This project was funded by the European Union and Span-ish Ministry for the Rural and Marine Environment (MARM)Project API 06-008 ‘‘Research into Colony Collapse Disorder(CCD) in Spain. Evaluation of epidemiological, environmental,and feeding factors’’.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jip.2012.06.008.

References

Alaux, C., Folschweiller, M., McDonnell, C., Beslay, D., Cousin, M., Dussaubat, C.,Brunet, J.L., Le Conte, Y., 2011. Pathological effects of the microsporidiumNosema ceranae on honey bee queen physiology (Apis mellifera). J. Invertebr.Pathol. 106 (3), 380–385.

Avery, S.W., Anthony, D.W., 1983. Ultrastructural study of early development ofNosema algerae in Anopheles albimanus. J. Invertebr. Pathol. 42, 87–95.

Bailey, L., 1955. The epidemiology and control of Nosema disease of the honey-bee.Ann. Appl. Biol. 43, 379–389.

Bailey, L., 1962. Bee diseases. Rep. Rothamstad exp. Stan. 1961, 16–161.Bomar, C.R., Lockwood, J.A., Pomerinke, M.A., French, J.D., 1993. Multiyear

evaluation of the effects of Nosema locustae (Microsporida: Nosematidae) onrangeland grasshoppers (Orthoptera: Acrididae) population density and naturalbiological controls. Environ. Entomol. 22 (2), 489–497.

Cali, A., 1991. General microsporidian features and recent findings on AIDS isolates.J. Protozool. 38, 625–630.

Chen, Y.P., Evans, J.D., Smith, J.B., Pettis, J.S., 2007. Nosema ceranae is a long-presentand widespread microsporidian infection of the European honey bee (Apismellifera) in the United States. J. Invertebr. Pathol. 92, 152–159.

Chen, Y., Evans, J.D., Smith, I.B., Pettis, J.S., 2008. Nosema ceranae is a long-presentand wide-spread microsporidean infection of the European honey bee (Apismellifera) in the United States. J. Invertebr. Pathol. 97, 186–188.

Chen, Y.P., Evans, J.D., Murphy, C.A., Gutell, R., Zuker, M., Gundersen-Rindal, D.E.,Pettis, J.S., 2009. Morphological, molecular, and phylogenetic characterizationof Nosema ceranae, a microsporidian parasite isolated from the European honeybee, Apis mellifera. J. Euk. Microb. 56, 142–147.

Cox-Foster, D.L., Conlan, S., Holmes, E., Palacios, G., Evans, J.D., Moran, N.A.,Quan, P.L., Briese, T., Hornig, M., Geiser, D.M., Martinson, V., van Engelsdorp,D., Kalkstein, A.L., Drysdale, A., Hui, J., Zhai, J., Cui, L., Hutchison, S.K.,Simons, J.F., Egholm, M., Pettis, J.S., Lipkin, W.I., 2007. A metagenomicsurvey of microbes in honey bee colony collapse disorder. Science 318, 283–287.

Dahle, B., 2010. The role of Varroa destructor for honey bee colony losses in Norway.J. Apic. Res. 49, 124–125.

Didier, E.S., 2005. Microsporidiosis: an emerging and opportunistic infection inhumans and animals. Acta Trop. 94, 61–76.

Dorrestein, G.M., van der Hage, M., 1999. A case of diarrhea caused byMicrosporidiosis in a 4-month-old blue fronted amazon parrot (Amazonaaestiva). Department of Pathology, Veterinary Faculty, Utrecht University.

Forsgren, E., Fries, I., 2010. Comparative virulence of Nosema ceranae and Nosemaapis in individual European honey bees. Vet. parasitol. 10, 1016.

Fries, I., Feng, F., Da Silva, A., Slemeda, S.B., Pieniazek, N.J., 1996. Nosema ceranae n.sp. (Microspora, Nosematidae), morphological and molecular characterizationof a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera,Apidae). Eur. J. Protistol. 32, 356–365.

Fries, I., Martín, R., Meana, A., García-Palencia, P., Higes, M., 2006. Natural infectionsof Nosema ceranae in European honey bees. J. Apic. Res. 45, 230–233.

Fries, I., 2010. Nosema ceranae in European honey bees (Apis mellifera). J. Invertebr.Pathol. 103, S73–S79.

General Register of Livestock Farms. Ministry of the Environment, Rural and MarineAffairs, 2008.

Guzman-Novoa, E., Eccles, L., Calvete, Y., McGowan, J., Kelly, P.G., Correa, A., 2010.Varroa destructor is the main culprit for the death and reduced populations ofoverwintered honey bee (Apis mellifera) colonies in Ontario, Canada. Apidologie.http://dx.doi.org/10.1051/apido/2009076.

Higes, M., Martin, R., Meana, A., 2006. Nosema ceranae, a new microsporidianparasite in honeybees in Europe. J. Invertebr. Pathol. 92, 93–95.

Higes, M., Garcia-Palencia, P., Martin-Hernandez, R., Meana, A., 2007. Experimentalinfection of Apis mellifera honeybees with Nosema ceranae (Microsporidia). J.Invertebr. Pathol. 94, 211–217.

Higes, M., Martin-Hernandez, R., Botias, C., Bailon, E.G., Gonzalez-Porto, A.V.,Barrios, L., Del Nozal, M.J., Bernal, J.L., Jimenez, J.J., Palencia, P.G., Meana, A.,2008. How natural infection by Nosema ceranae causes honeybee colonycollapse. Environ. Microbiol. 10, 2659–2669.

Higes, M., Martín-Hernández, R., Martínez-Salvador, A., Garrido-Bailón, E.,González-Porto, A.V., Meana, A., Bernal, J.L., Del Nozal, M.J., Bernal, J., 2009a. Apreliminary study of the epidemiological factors related to honey bee colonyloss in Spain. Environ. Microbiol. 10, 1758–2229.

Higes, M., Martín-Hernández, R., Garrido-Bailón, E., González-Porto, A.V., García-Palencia, P., Meana, A., Nozal, M.J.D., Mayo, R., Bernal, J.L., 2009b. Honeybeecolony collapse due to Nosema ceranae in professional apiaries. Environ.Microbiol. Rep. 1, 110–113.

Invernizzia, C., Abuda, C., Tomascoa, I., Harriet, J., Ramalloc, G., Campáb, J., Katzb, H.,Gardiolb, G., Mendozac, Y., 2009. Presence of Nosema ceranae in honeybees (Apismellifera) in Uruguay. J. Invertebr. Pathol. 101, 150–153.

Jacobson, E.R., Green, D.E., Undeen, A.H., Granfield, M., Vaughn, K.L., 1998. SystemicMicrosporidiosis in Inland Bearded Dragons (Pogona vitticeps). J. Zoo. WildlifeMed. 29 (3), 315–323.

Klee, J., Besana, A.M., Genersch, E., Gisder, S., Nanetti, A., Tam, D.Q., Chinh, T.X.,Puerta, F., Ruz, J.M., Kryger, P., Message, D., Hatjina, F., Korpela, S., Fries, I.,Paxton, R.J., 2007. Widespread dispersal of the microsporidian Nosema ceranae,an emergent pathogen of the western honey bee, Apis mellifera. J. Invertebr.Pathol. 96, 1–10.

Kudo, R.R., 1954. Protozoology. Charles C. Thomas.Lee, S.C., Corradi, N., Byrnes III, E.J., Torres-Martinez, S., Dietrich, F.S., Keeling, P.J.,

Heitman, J., 2008. Microsporidia evolved from ancestral sexual fungi. Curr. Biol.18, 1675–1679.

Le Conte, Y., Ellis, M., Ritter, W., 2010. Varroa mites and honey bee health: canVarroa explain part of the colony losses? Apidologie 41, 353–363.

Lom, J., Nilsen, F., 2003. Fish microsporidia: fine structural diversity and phylogeny.Int. J. Parasitol. 33, 107–127.

Liu, T.P., 1984. Ultrastructure of the midgut of the worker honey bee Apis melliferaheavily infected with Nosema apis. J. Invertebr. Pathol. 44, 282–291.

Liu, F., Wang, Q., Dai, P.L., Wu, Y.Y., Song, H.K., Zhou, T., 2008. Natural stripeof microsporidia of honeybee in China, Chinese Bull. Entomolology 45,963–966.

Matheson, A., 1996. World bee health update. Bee World 77, 45–51.Martin-Hernandez, R., Meana, A., Prieto, L., Salvador, A.M., Garrido-Bailon, E., Higes,

M. 2007. The outcome of the colonization of Apis mellifera by Nosema ceranae.Appl. Environ. Microbiol. AEM.00270-00207.

110 J.M. Fernández et al. / Journal of Invertebrate Pathology 111 (2012) 106–110

Paxton, R.J., Klee, J., Korpela, S., Fries, I., 2007. Nosema ceranae has infected Apismellifera in Europe since at least 1998 and may be more virulent than Nosemaapis. Apidologie 38, 1–9.

Paxton, R.J., 2010. Does infection by Nosema ceranae cause ‘‘Colony CollapseDisorder’’ in honey bees (Apis mellifera)? J. Apic. Res. 49, 80–84.

Percy, D.H., Barthold, S.W., 1993. Pathology of Laboratory Rodents and Rabbits. IowaState University Press, Ames IA, pp. 210–214.

Puerta, F., Flores, J.M., Ruíz, J.A., Ruz, J.M., Campano, F. 2001. Enfermedades de lasabejas. Prevención, diagnóstico y tratamiento. (Eds.), COAG – Andalucía.Consejería de Agricultura y Pesca. Junta de Andalucía.

Shadduck, J.A., Meccoli, R.A., Davis, R., Font, R.L., 1990. First isolation of amicrosporidian from a human patient. J. Infect. Dis. 162, 773–776.

Siede, R., Berg, S., Meixner, M. 2008. Effects of symptomless infections with Nosemasp. on honey bee colonies. In: OIE – Symposium Diagnosis and Control of BeeDiseases, August 26–28. Freiburg, Germany.

Traver, B.E., Fell, R.D., 2011. Nosema ceranae in drone honey bees (Apis mellifera). J.Invertebr. Pathol. 107, 234–236.

Wasson, K., Peper, R.L., 2000. Mammalian microsporidiosis. Vet. Pathol. Mar. 37 (2),113–128.

Williams, G.R., Shafer, A.B.A., Rogers, R.E.L., Shutler, D., Stewart, D.T., 2008. Firstdetection of Nosema ceranae, a microsporidean parasite of European honeybees (Apis mellifera), in Canada and central USA. J. Invertebr. Pathol. 97,189–192.