Low prevalence of honeybee viruses in Spain during 2006 and 2007.
ABSTRACT RNA viruses that affect honeybees have been involved in colony losses reported around the world. The aim of the present work was to evaluate the prevalence and distribution of honeybee viruses during 2006-2007 in Spanish professional apiaries, and their association with colony losses. Four hundred and fifty-six samples from apiaries located in different geographic regions of Spain were analyzed. Thirty-seven percent of the samples had viral presence. Most (80%) had one virus and 20% two different viruses. All the analyzed viruses, Deformed Wing Virus (DWV), Israeli Acute Paralysis Virus (IAPV), Black Queen Cell Virus (BQCV), Sacbrood Virus (SBV) and Kashmir Bee Virus (KBV) were detected, but detection rates were lower than expected. According to these results and considering the high prevalence of other honeybee pathogens in Spain, the role of viruses in colony losses in Spain may be discussed.
- SourceAvailable from: Dennis Vanengelsdorp[show abstract] [hide abstract]
ABSTRACT: The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD), which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus with no DNA stage placed taxonomically within the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNA interference technology (RNAi) has been used successfully to silence endogenous insect (including honey bee) genes both by injection and feeding. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory conditions. In the current study IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This controlled study included a total of 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). To our knowledge, this is the first successful large-scale real world use of RNAi for disease control.PLoS Pathogens 01/2010; 6(12):e1001160. · 8.14 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: In recent years, a worldwide decline in the Apis mellifera populations has been detected in many regions, including Spain. This decline is thought to be related to the effects of pathogens or pesticides, although to what extent these factors are implicated is still not clear. In this study, we estimated the prevalence of honey bee colony depopulation symptoms in a random selected sample (n = 61) and we explored the implication of different pathogens, pesticides and the flora visited in the area under study. The prevalence of colony depopulation symptoms in the professional apiaries studied was 67.2% [95% confidence interval (CI) = 54.6-79.8; P < 0.0001]. The most prevalent pathogen found in the worker honey bee samples was Nosema ceranae[65.6%; 95% CI = 52.8-78.3; P < 0.0001], followed by Varroa destructor[32.7%; 95% CI = 20.2-45.4; P < 0.0001] and 97.5% of the colonies infected by N. ceranae were unhealthy (depopulated). Co-infection by V. destructor and N. ceranae was evident in 22.9% (95% CI = 11.6-34.3; P < 0.0001) of the samples and only in unhealthy colonies. Of the 40 pesticides studied, only nine were detected in 49% of the stored pollen samples analysed. Fipronil was detected in only three of 61 stored pollen samples and imidacloprid was not detected in any. Acaricides like fluvalinate, and chlorfenvinphos used to control Varroa mite were the most predominant residues in the stored pollen, probably as a result of their application in homemade formulae. None of the pesticides identified were statistically associated to colony depopulated. This preliminary study of epidemiological factors suggests that N. ceranae is a key factor in the colony losses detected over recent years in Spain. However, more detailed studies that permit subgroup analyses will be necessary to contrast these findings.Environmental Microbiology Reports 04/2010; 2(2):243-50. · 2.71 Impact Factor
- Bee World. 01/1996; 77.
Low prevalence of honeybee viruses in Spain during 2006 and 2007
K. Antúneza,⇑, M. Anidoa, E. Garrido-Bailónb, C. Botíasb, P. Zuninoa, A. Martínez-Salvadorc,
R. Martín-Hernándezb, M. Higesb
aDepartamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay
bLaboratorio de Patología Apícola, Centro Apícola Regional, JCCM, Marchamalo, Spain
cConsultor epidemiólogo, Madrid, Spain
a r t i c l ei n f o
Received 26 August 2011
Accepted 16 March 2012
a b s t r a c t
RNA viruses that affect honeybees have been involved in colony losses reported around the world. The
aim of the present work was to evaluate the prevalence and distribution of honeybee viruses during
2006–2007 in Spanish professional apiaries, and their association with colony losses. Four hundred
and fifty-six samples from apiaries located in different geographic regions of Spain were analyzed.
Thirty-seven percent of the samples had viral presence. Most (80%) had one virus and 20% two different
viruses. All the analyzed viruses, Deformed Wing Virus (DWV), Israeli Acute Paralysis Virus (IAPV), Black
Queen Cell Virus (BQCV), Sacbrood Virus (SBV) and Kashmir Bee Virus (KBV) were detected, but detection
rates were lower than expected. According to these results and considering the high prevalence of other
honeybee pathogens in Spain, the role of viruses in colony losses in Spain may be discussed.
? 2012 Elsevier Ltd. All rights reserved.
Honeybees play an essential role in the ecology of natural envi-
ronments and in agricultural production through pollination. The
value of honeybee pollination for agriculture has been estimated
at more than $14.6 billion in the United States and $443 million
in Canada (Delaplane and Mayer, 2000; Morse and Calderone,
2000). According to FAO and the European Union, the value of pol-
lination is 20–30 times higher than the value of honey production.
However, honeybees are susceptible to a variety of pathogens
(Morse and Flottum, 1997; Schmid-Hempel, 1998), such as the
mites Varroa destructor and Acarapis woodi, the microsporidia
Nosema ceranae and Nosema apis, the bacteria Paenibacillus larvae
and Melissococcus plutonius and RNA viruses. More than 18 RNA
viruses that affect honeybees have been described. Most of them
cause unapparent infections without clinical signs but in certain
cases may cause serious or lethal diseases (Allen and Ball, 1996;
Chen and Siede, 2007). Black Queen Cell Virus (BQCV), Deformed
Wing Virus (DWV), Sacbrood Virus (SBV), Chronic Bee Paralysis
Virus (CBPV), Acute Bee Paralysis Virus (ABPV), Kashmir Bee Virus
(KBV), and Israeli Acute Paralysis Virus (IAPV) (Chen and Siede,
2007; Maori et al., 2007; de Miranda et al., 2010; de Miranda and
Genersch, 2010; Ribière et al., 2010) may be considered among
the most important viruses that affect honeybees.
BQCV was first detected in queen larvae and prepupae that
turned brown to black (Bailey and Woods, 1974). However, it also
affects larvae and pupae of worker bees without causing signs.
DWV is one of the most studied viruses that affect honeybees
due to its relation with colony losses induced by V. destructor.
Co-infection of pupae with both pathogens causes clinical signs,
such us pupal death and adult bees with deformed wings and
shortened abdomen which dies soon after emergence (de Miranda
and Genersch, 2010).
SBV affects larvae of honeybees that acquire a pale yellow color;
their skin become leathery and the ecdysial fluid accumulates be-
tween the body and the skin (Ball and Bailey, 1997).
ABPV, KBV and IAPV are part of a complex of related viruses. At
low levels, infected colonies do not show clinical signs, but at high
levels of virus challenge, there are high mortality rates. These
viruses have been associated with honeybee colony losses, espe-
cially when colonies are co-infected with V. destructor (Cox-Foster
et al., 2007; Maori et al., 2007; de Miranda et al., 2010).
RNA viruses have been associated with the occurrence of mas-
sive honeybee colony losses around the world, especially in the
northern hemisphere countries (Cox-Foster et al., 2007; Genersch
and Aubert, 2010; Highfield et al., 2009). Although in Spain there
is no official information about the extent of colony losses, accord-
ing to the information from the service of diagnosis of the Bee
Pathology Laboratory, Centro Apicola Regional during 2006 and
2007, between 30 and 40% of the colonies died during winter
The aim of the present work was to evaluate the prevalence and
distribution of different honeybee viruses during 2006–2007 in
0034-5288/$ - see front matter ? 2012 Elsevier Ltd. All rights reserved.
⇑Corresponding author. Address: Departamento de Microbiología, Instituto de
Investigaciones Biológicas Clemente Estable, Avda., Italia 3318, CP 11600, Monte-
video, Uruguay. Tel.: +598 2487 1616; fax: +598 2487 5548.
E-mail addresses: email@example.com, firstname.lastname@example.org (K. Antúnez).
Research in Veterinary Science 93 (2012) 1441–1445
Contents lists available at SciVerse ScienceDirect
Research in Veterinary Science
journal homepage: www.elsevier.com/locate/rvsc
Spanish professional apiaries and discuss their possible association
with colony losses episodes reported during these years. Although
this information is not updated, during 2006 and 2007 high epi-
sodes of colony losses were reported in this country. Other works
carried out during the same period indicated that N. ceranae was
the causative agent of colony losses in Spain during these years
(Higes et al., 2006, 2008; Martin-Hernandez et al., 2007), although
the role of bee viruses had not been deeply studied. Moreover, this
study will be useful to compare results obtained in further surveys
carried out to elucidate the factors responsible for massive colony
2. Materials and methods
2.1. Honeybee samples
The present study was part of a wider survey designed to eval-
uate the factors associated with a ‘‘high colony losses phenome-
non’’ in Spain, and included the evaluation of many different
pathogens and pesticides in worker bees and pollen. Two surveys
were carried out during spring and autumn of 2006 and 2007,
and in the whole country (Fig. 1). In each region of Spain apiaries
were randomly selected and one colony per apiary was randomly
selected for the analysis.
Adult worker honeybees were collected from frames, trans-
ported alive to the laboratory and maintained at ?80 ?C until anal-
ysis in order to avoid RNA degradation (Chen and Siede, 2007).
Four hundred and fifty-six samples were used for virus analysis.
From these, 287 were collected in the spring and 68 in autumn of
2006, and 69 in spring and 32 in autumn of 2007.
2.2. Sample processing
Ten adult honeybees were randomly selected from each sample
and transferred aseptically to sterile plastic bags and 5 ml of cold
50% AL buffer containing RNA carrier (Qiagen) were added. Bees
were crushed for 2 min at high speed in a Stomacher blender and
the resultant homogenates were centrifuged at 1500g for 10 min
at 4??C. 400 lL of the resulting supernatants were recovered and
used for total nucleic acid extraction.
2.3. Nucleic acid extraction, genomic DNA degradation and cDNA
All reactions were performed in 96-well microtitre plates. Four
hundred microliter of the honeybee supernatants were incubated
with protease (Qiagen) and incubated at 70 ?C for 15 min. Then,
supernatants were subjected to nucleic acid extraction using Bio-
sprint 96 DNA Blood (Qiagen) and BioSprint workstation (Qiagen),
using the Tissue program.
Obtained nucleic acids were then subjected to DNA digestion
with DNase I (Qiagen) in order to completely remove genomic
DNA. The total recovered RNA was immediately used to generate
first strand cDNAs using the Quantitec Reverse Transcription Kit
(Qiagen), according to the manufacturer’s instructions. Negative
and positive controls were run in parallel for each step (nucleic
acid extraction and reverse transcription reactions).
All real-time quantitative PCR reactions were carried out in 96-
well microtiter plates using the Fast Start DNA Master Plus SYBR
Green I (Roche) and LightCycler 480 Real Time PCR System (Roche).
Specific primers for the amplification of b-actin mRNA and dif-
ferent honeybee viruses (BQCV, DWV, KBV, IAPV and SBV) were
used. Primer sequences and references are shown in Table 1. In
order to evaluate the RNA quality of the different samples, the gene
that encodes for honeybee b-actin was amplified and only those
samples that were positive for b-actin (which indicated a good le-
vel of RNA conservation) were analyzed for the presence of viruses.
The reaction mixture consisted of 1? Light Cycler Fast Start
Master Plus Reaction Mix SYBR Green containing Light Cycler Fast
Fig. 1. Geographical distribution of samples in, during spring and autumn of 2006 and 2007.
K. Antúnez et al./Research in Veterinary Science 93 (2012) 1441–1445
Start enzyme, 0.5 lM of each primer (one pair of primers per reac-
tion), RNAse-free water and 5 lL of 1:10 diluted cDNA in a final
volume of 20 lL. Negative controls were carried out excluding nu-
cleic acids from the reaction.
The cycling program consisted of an initial preincubation step of
95 ?C for 10 min, and 40 cycles of 95 ?C for 10 s, 50 ?C/60 ?C for 10 s
and 72 ?C for 14 s. For amplification of b-actin, 50 ?C was used as
the annealing temperature, while for virus analysis a 60 ?C temper-
ature was used.
Fluorescence was measured in the elongation step and negative
controls (without DNA) were included in each reaction run. The
specificity of the reaction was checked by the analysis of the melt-
ing curve of the final amplified product, which was obtained
through continuous reading over increasing temperatures from
65 ?C to 95 ?C (5 readings at each ?C).
The amplification results from the different viruses and b-actin
are shown by the software as the threshold cycle (Ct) value, which
represented the number of cycles needed to generate a fluorescent
signal greater than a predefined threshold. Data of presence or ab-
sence of each virus per sample was used to calculate prevalence of
each virus in the total sampling or by season.
Honeybee samples were subjected to genomic DNA degrada-
tion, retrotranscription and qRT-PCR in order to detect the pres-
ence of b-actin mRNA and different viruses. Correct amplification
of b-actin mRNA confirmed the conservation of RNA and the suit-
ability of the samples for viral analysis. Amplified fragments for
different viruses presented the expected sizes and identification
was confirmed by sequencing and melting temperature analysis
Viral detection rates were low, since only 36.6% of the samples
were affected. Most of the affected samples (80%) contained one
virus and 20% contained two. Only in one sample obtained in
2006 and another in 2007 were detected three viruses (KBV–
IAPV–DWV) in spring.
During 2006 the most prevalent virus was DWV (18.6%) (Table
2A), with a significant higher prevalence in autumn compared to
spring (v2, p < 0.0001) (Table 2B). IAPV was the second most preva-
lent virus (13.0%) and also presented a significant higher presence
in autumn (v2, p < 0.0001) (Table 2B). BQCV was the third most fre-
quently identified virus (10.4%), followed by SBV (1.1%) and KBV
(0.3%) were the least prevalent viruses. Taking into account mixed
infections, BQCV–DWV was more prevalent in spring (4.2%) and
IAPV–DWV in autumn (10.3%) (Table 2B).
During 2007, the most prevalent virus was IAPV (25.7%). BQCV
the second more prevalent virus (9.9%) and was followed by DWV
(5.9%). KBV was only detected in one sample (1.0%) and SBV was
not detected. In this year, IAPV was more prevalent in spring than
in autumn but this difference was not significant (v2, p > 0.05). For
BQCV we recorded the same situation (v2, p > 0.05). Curiously,
DWV was only detected in spring samples (Table 2C).
In this year, mixed infection was only detected during spring
(Table 2C) with the same prevalence in all cases, while the co-
infection BQCV–IAPV was detected only in spring. In both years,
KBV was only detected during spring, while IAPV presented a high-
er infection rate in autumn, but this difference was only significant
in 2006 (v2, p < 0.0001) but not in 2007 (v2, p > 0.05) (Tables 2B
Comparing the total results obtained in 2006 and 2007 (Table
2A), DWV presented a significantly higher prevalence in 2006
(v2, p < 0.01) and IAPV in 2007 (v2, p < 0.01). IAPV and DWV co-
infection was more common in 2007 (v2, p < 0.001). The other
viruses or combinations did not show significant prevalence differ-
ences in any year.
The method used in the present work for detection of honeybee
viruses was a rapid, confident and easy way for the simultaneous
analysis of several honeybee samples and was useful for large scale
Honeybee viral prevalence found in Spain was surprisingly lower
than expected when compared with the situation observed in other
countries, as suggested in preliminarystudies (Garrido-Bailón et al.,
2010; Higes et al., 2008).
DWV is one of the most prevalent viruses around the world. In
Hungary, it has been reported to have a prevalence of 72%, 97% in
France, 91% in Austria, 97% in England and 100% in Uruguay (Tent-
cheva et al., 2004; Antunez et al., 2006; Berenyi et al., 2006; Baker
and Schroeder, 2008; Forgach et al., 2008). However, in the present
study only 18.6 and 5.9% of Spanish samples were affected during
2006 and 2007, respectively.
BQCV has been reported to have a variable prevalence in differ-
ent countries, with 54, 86, 30 and 90% detection rates in Hungary,
France, Austria and Uruguay, respectively (Antunez et al., 2006;
Berenyi et al., 2006; Forgach et al., 2008; Tentcheva et al., 2004).
In Spain, the detection rate was 10.4% in 2006 and 9.9% in 2007.
SBV detection rate was low in the collection of Spanish samples
assessed in this study (only 1.1% detected in 2006). These values
were similar to others reported in Hungary (2%) and England
(1.4%) (Baker and Schroeder, 2008; Forgach et al., 2008). However,
detection rates in France, Austria and Uruguay were higher, being
86, 49 and 100%, respectively (Antunez et al., 2006; Berenyi et al.,
2006; Tentcheva et al., 2004).
So far, KBV has not been detected in Hungary, Austria or Uru-
guay (Antunez et al., 2006; Berenyi et al., 2006; Forgach et al.,
Primers used for the detection of honeybee viruses.
Virus Primer Sequence 50–30
ReferenceFragment sizeTm (?C) Efficiency Detection limit
Kukielka et al. (2008a)30584nd nd
DWVKukielka et al. (2008a) 250 84.587.5%1 ? 10?7
KBVJohnson et al. (2009) 127 80.5 115%1 ? 10?7
SBVJohnson et al. (2009) 10580.588.8%1 ? 10?5
IAPVPalacios et al. (2008) 114 8196.8%1 ? 10?7
BACTIN Yang and Cox-Foster (2007)156 82.5 110.9%1 ? 10?5
nd, not determined.
K. Antúnez et al./Research in Veterinary Science 93 (2012) 1441–1445
2008). In Spain, its detection rate was 0.3% in 2006 and 1.0% in
2007, while in France 17% of affected samples were detected
(Tentcheva et al., 2004).
Lastly, IAPV showed a prevalence of 13.0% in 2006 and 25.7% in
2007, in agreement with a preliminary study carried out in Spain
which reported 18% of samples in which the virus was detected
(Garrido-Bailón et al., 2010). A similar detection rate (14%) was
determined in France (Blanchard et al., 2008). Although this virus
has been associated with colony losses in the United States (Cox-
Foster et al., 2007; Hunter et al., 2010), the low prevalence found
in France and Spain indicates that it would not be responsible for
the massive colony losses detected in these countries in the last
The results of the present work contrast with those previously
reported by Kukielka et al. (2008b) obtained from Spanish samples
collected during 2004–2006. A possible reason is that samples used
by Kukielka et al. (2008b) were not randomly selected; these sam-
ples belong to the diagnosis service from the Laboratorio de Pato-
logía Apícola (Centro Apícola Regional, JCCM, Marchamalo, Spain)
and were submitted by beekeepers who suspected the presence
of pathogens or belonged to collapsing colonies. Seventy percent
of the sampled colonies presented a wide range of clinical signs
compatible with infection by at least one of the viral diseases, such
as bloated abdomens, disorientation, and weakness and depopula-
tion of the colony. This might explain the high viral infection levels
In the present work, apiaries and samples were randomly se-
lected from around the country independently of the sanitary situ-
ation of the colonies. This is the appropriate way to assess the real
prevalence of a pathogen in a geographical area. In previous sur-
veys such us those carried out in Hungary, Austria, Uruguay, or
even Spain, most of the samples were collected after the sudden
collapse of colonies and so the real prevalence of honeybee viruses
in these cases was overestimated (Antunez et al., 2005, 2006;
Berenyi et al., 2006; Forgach et al., 2008; Kukielka et al., 2008b).
It is possible that the higher viral titers found in collapsed colonies
in other countries were a consequence of colony weakness, debil-
itated by the presence of other pathogens, or that viruses were
present in a latent state, or simply due to a different types of sam-
ples analyzed (in unhealthy colonies the viral prevalence may be
On the other side, in the present work bees were collected from
frames comprised of young bees with lower infection levels; com-
pared to those analyzed by other authors (Antunez et al., 2005,
2006; Berenyi et al., 2006; Forgach et al., 2008; Kukielka et al.,
It is important to mention that ABPV was also included in the
present analysis, using the primers ACCGACAAAGGGTATGATGC
and CTTGAGTTTGCGGTGTTCCT, reported by Johnson et al., 2009.
However, the results were confusing (Tm of different amplicons
varied between 82 and 90 ?C) and we were not able to sequence
all suspected amplicons and so decided not to include those results.
Prevalence and co-infection rates of honeybee viruses in Spanish professional apiaries during 2006 and 2007, evaluated by qRT-PCR. A: for each year, B and C: for every year and
period of sampling. Bold indicates statistical significant results.
Viruses Total 2006Total 2007 Interannual comparison
% CI (95%)% CI (95%)
Spring 06Autumn 06 Interseasonal comparison
Spring 07 Autumn 07Interseasonal comparison
K. Antúnez et al./Research in Veterinary Science 93 (2012) 1441–1445
Considering the low prevalence of RNA viruses detected in the
present work and the high prevalence of other honeybee patho-
gens in different regions of Spain, such as N. ceranae or V. destructor
(Bernal et al., 2010; Higes et al., 2010; Martin-Hernandez et al.,
2007), it might be suggested that the studied viruses would not ex-
plain by themselves, the generalized colony loss phenomenon in
Although DWV, IAPV, BQCV, SBV and KBV were detected in
samples from different geographic areas of Spain during 2006
and 2007, viral detection rates were low, considering that during
those years between 30 and 40% of the Spanish colonies died, it
might be suggested that other causes might have a role in the col-
ony losses in Spain during 2006 and 2007.
Conflicts of interest statement
There are no conflicts of interest to be declared.
The authors wish to thank the Spanish beekeeper Associations
and professional beekeepers for supplying the samples. We would
also like to thank Almudena Cepero, Virginia Albendea, Carmen
Abascal, Carmen Rogerio and Teresa Corrales for their technical
This study was supported by the Junta de Comunidades de
Castilla-La Mancha (Consejería de Agricultura and Consejería de
Educación), INIA-FEDER funds (RTA2005-152, RTA 2008-00020-
C02-01 and RZ00-013) and MARM-FEAGA funds (Programa Nac-
ional Apícola 2011–2013).
K. Antúnez, M. Anido and P. Zunino international travel grants
were covered by Agencia Nacional de Investigación e Innovación
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