Nosema ceranae is a long-present and wide-spread microsporidian infection of the European honey bee (Apis mellifera) in the United States.
ABSTRACT Honey bee samples collected between 1995 and 2007 from 12 states were examined for the presence of Nosema infections. Our results showed that Nosema ceranae is a wide-spread infection of the European honey bee, Apis mellifera in the United States. The discovery of N. ceranae in bees collected a decade ago indicates that N. ceranae was transferred from its original host, Apis cerana to A. mellifera earlier than previously recognized. The spread of N. ceranae infection in A. mellifera warrants further epidemiological studies to identify conditions that resulted in such a widespread infection.
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ABSTRACT: To determine whether the introduction of Carniolan stock may alter the impact of Nosema apis on New Zealand bees, the responses of Italian (Apis mellifera ligustica) and dark (Apis mellifera mellifera) New Zealand bees and Carniolan (Apis mellifera carnica) bees from Australia to dosing with N apis spores were compared. Newly emerged adult bees were individually dosed with 2 x 105 N apis spores, caged together in groups of 50, and incubated at 33°C. The longevity of each bee and number of N apis spores carried by each bee at the time of death were recorded. All bees dosed with N apis spores had significantly reduced longevity compared with the undosed control bees. Furthermore, there were no significant differences among the 3 stocks of bees in the degree of this reduction in longevity. However, dark and Carniolan bees survived better in cages than Italian bees, whether dosed or not. There were significant differences among the 3 stocks in the mean numbers of spores carried by each dosed bee at the time of death, with Italian bees carrying the highest spore loads, Carniolan bees the lowest, and dark bees carrying an intermediate number of spores. Thus, Carniolan bees from Australia may support a slower rate of N apis proliferation and thus have lighter infections than New Zealand dark or Italian bees receiving similar doses of spores.http://dx.doi.org/10.1051/apido:19950606. 01/1995;
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ABSTRACT: The economically most important honey bee species, Apis mellifera, was formerly considered to be parasitized by one microsporidian, Nosema apis. Recently, [Higes, M., Martín, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93-95] and [Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A Nosema ceranae isolate from the honeybee Apis mellifera. Apidologie 38, 30-37] used 16S (SSU) rRNA gene sequences to demonstrate the presence of Nosema ceranae in A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between N. apis and N. ceranae based on PCR-RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (N =9 isolates gave N. apis RFLPs and sequences, N =20 isolates gave N. ceranae RFLPs and sequences; 100% correct classification). We then employed the method to analyze N =115 isolates from across the world. Our data, combined with N =36 additional published sequences demonstrate that (i) N. ceranae most likely jumped host to A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii) N. ceranae is now a parasite of A. mellifera across most of the world. The rapid, long-distance dispersal of N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping.Journal of Invertebrate Pathology 09/2007; 96(1):1-10. · 2.67 Impact Factor
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ABSTRACT: Based on light microscopic and ultrastructural characteristics as well as on the nucleotide sequence of the small subunit ribosomal RNA coding region, the microsporidium Nosema ceranae n. sp., a parasite of the Asian honey bee Apis cerana is described. Merogonial stages and sporonts are diplokaryotic. Merozoites are mostly formed by cytoplasmic fission in quadrinucleate meronts and the number of merogonial cycles may vary. The sporogony is disporoblastic. The living mature spore is ovocylindrical, straight to slightly curved and measures 4.7 × 2.7 μm whereas fixed and stained spores measure 3.6 × 1.7 μm. The polar filament is isofilar with a diameter of 96–102 nm and is arranged in 20–23 coils in the posterior and mid-part of the spore. In the anterior part of the polaroplast there are closely packed approximately 11 nm thick lamellae. The lamellae of the posterior polaroplast are thicker and less regular. In the posterior part of the mature spore a well fixed posterior body interpreted as a posterosome was often observed. Phylogenetic analysis, based on the sequence of the small subunit ribosomal RNA, places Nosema ceranae in the Nosema clade, as defined by Nosema bombycis, the type species of the Nosema genus.European Journal of Protistology - EUR J PROTISTOL. 01/1996; 32(3):356-365.
Nosema ceranae is a long-present and wide-spread
microsporidian infection of the European honey bee
(Apis mellifera) in the United States
Yanping Chen, Jay D. Evans, I. Bart Smith, Jeffery S. Pettis*
USDA-ARS Bee Research Laboratory, Bldg. 476, BARC-East, Beltsville, MD 20705, USA
Received 6 April 2007; accepted 23 July 2007
Available online 6 August 2007
Honey bee samples collected between 1995 and 2007 from 12 states were examined for the presence of Nosema infections. Our results
showed that Nosema ceranae is a wide-spread infection of the European honey bee, Apis mellifera in the United States. The discovery of
N. ceranae in bees collected a decade ago indicates that N. ceranae was transferred from its original host, Apis cerana to A. mellifera
earlier than previously recognized. The spread of N. ceranae infection in A. mellifera warrants further epidemiological studies to identify
conditions that resulted in such a widespread infection.
Published by Elsevier Inc.
Keywords: Nosema disease; Honey bee; Apis mellifera; Apis cerana; Nosema ceranae; Nosema apis; rRNA
Nosema disease is one of the most prevalent adult honey
bee diseases (Bailey, 1981; Matheson, 1993) and is caused
by two described species of microsporidia, Nosema apis
and Nosema ceranae. Transmission of Nosema in honey
bee colonies is mainly via the fecal-oral route in which
pathogens are spread by transferring feces of diseased hosts
to uninfected hosts via ingestion. Adult bees ingest Nosema
spores when they are eating contaminated food and when
they are cleaning up fecal material from infected bees.
The spores geminate within the midgut and release polar
tubes that transfer their sporoplasm into midgut epithelial
cells where they generate more spores. Millions of new
spores can be found inside of a bee’s midgut a few weeks
after initial infection (Bailey and Ball, 1991) and the spores
excreted with feces become new sources of the infection in
the colonies. Although infected bees do not exhibit obvious
external disease symptoms, infection of Nosema causes
digestive disorders, shortened life spans of honey bees,
decrease of population size of honey bee colonies (Hassan-
ein, 1953; Rinderer and Sylvester, 1978; Malone et al.,
1995), and reduction of honey production and crop prod-
ucts that rely on bees for pollination (Anderson and Gia-
con, 1992; Fries et al., 1984; Goodwin et al., 1990).
Since late 2006, a mysterious illness, termed ‘‘Colony
Collapse Disorder’’ (CCD), has been devastating massive
numbers of honey bee colonies and threatens the beekeep-
ing industry in the United States. As part of our efforts to
identify the causes of sudden disappearances and extensive
die-offs of colonies in the hope of designing appropriate
control strategies to safeguard bee health in the future,
the status of Nosema infections in honey bees collected from
states affected by CCD was also examined. While our inves-
tigation did not show a direct correlation between Nosema
infection and CCD, the results revealed that N. ceranae, a
species of Nosema originally found in the Asian honey
bee, Apis cerana (Fries et al., 1996), was the only Nosema
species found in infected European honey bees (Apis melllif-
era) and had a wide geographical distribution in the United
States. For years, Nosema disease of the European honey
bee was exclusively attributed to N. apis. However, two
recent reports showed that N. ceranae has been discovered
0022-2011/$ - see front matter Published by Elsevier Inc.
*Corresponding author. Fax: +1 301 504 8736.
E-mail address: Jeff.Pettis@ars.usda.gov (J.S. Pettis).
Available online at www.sciencedirect.com
Journal of Invertebrate Pathology 97 (2008) 186–188
in the European honey bee in Taiwan and Spain (Higes
et al., 2006; Huang et al., 2007). In addition, Klee et al.
(2007) reported that N. ceranae was a widespread infection
of the European honey bee worldwide. In light of the dis-
covery that N. ceranae is a widespread infection of Euro-
pean honey bees, we hypothesized that N. ceranae might
have been present in the European honey bees in the United
States long before we realized. To clarify this, honey bee
samples collected a decade ago were examined for the pres-
ence of N. ceranae. Here we present evidence that N. cer-
anae is a long-established and wide-spread infection of the
European honey bee in the United States.
Bee samples were collected from the field and sent to our
laboratory by beekeepers. The samples came from different
geographic regions of the United States from the years
1995–2007. All the bee samples were stored in 70% ethanol
at room temperature prior to examination. Ten bees were
randomly selected from each group and a total 180 bees
were examined for the presence of Nosema. Genomic
DNA was extracted from individual bees using a DNA
purification kit, DNAzol (Invitrogen, Carlsbad, CA)
according to the manufacturer’s protocol. The complete
sequences of rRNA including small subunit ribosomal
RNA gene, internal transcribed spacer, and large subunit
ribosomal RNA gene, for both N. apis (GenBank Acces-
sion No: U97150) and N. ceranae (GenBank Accession
No: DQ486027) were retrieved from GenBank and aligned
with the MegAlign (DNASTAR Lasergene software pro-
gram, Madison, WI). Based on the alignment of rRNA, a
set of generic primers, Nosema F (50-ggcagttatgggaagta-
aca-30)/Nosema R (50-ggtcgtcacatttcatctct-30) was designed
in the region conserved in both N. apis and N. ceranae to
allow for amplification of rRNA in both species. To differ-
entiate between two the Nosema species, one set of primers,
N. ceranae F (50-cggataaaagagtccgttacc-30)/N. ceranae R
(50-tgagcagggttctagggat-30) was designed in the region com-
plimentary only to N. ceranae, while another set of primers,
N. apis F (50-ccattgccggataagagagt-30/N. apis R (50-cac-
gcattgctgcatcattgac-30) was designed in the region compli-
mentary only to N. apis. DNA from individual bees was
amplified with three sets of Nosema primers separately.
Invitrogen recombinant TaqDNA polymerase was used
for PCR amplification and the reaction mixture as well as
PCR thermal cycling profiles was performed according to
the manufacturer’s recommended protocol. The specificity
of PCR amplification of each pair of primers was con-
firmed by purifying PCR fragments from agarose gels using
Wizard PCR Prep DNA Purification System (Promega,
Madison, WI), sequencing the PCR fragments, and com-
paring sequence results with sequences deposited in the
GenBank database, NCBI, NIH. DNA extracted from
Nosema infected bees (provided by Dr. Stephen F. Pernal
from Agriculture Agri-Food Canada) that were confirmed
to be positive for both N. ceranae and N. apis by PCR
method and sequencing analysis was used as a positive con-
trol. Negative controls (H2O) were also included in each
run of PCR amplification.
Our results showed that N. ceranae is the only Nosema
species found to infect European honey bees from our
widespread geographic collections in the United States.
N. ceranae infected bees were found in samples collected
from each of 12 states including Oregon, California,
Hawaii, Idaho, North Dakota, Minnesota, Texas, Ohio,
Tennessee, Connecticut, Maryland and Florida, represent-
ing the Northeast, Southeast, Midwest, Southwest, and the
West regions of the U.S. Among 180 bees examined for
Nosema, 16% of the bees (28/180) were positive for N. cer-
anae. The results of PCR amplification with generic
Nosema primer pair perfectly matched the results of ampli-
fication with specific N. ceranae primer pair. However, the
results showed that all examined bees were negative for N.
apis when amplified with specific N. apis primer pair.
In an attempt to investigate the historical incidence of
N. ceranae infection in European honey bees, we found
infections of N. ceranae in samples collected during the per-
iod from 1995 to 2007 in the United States (Fig. 1). The
detection of N. ceranae in bees collected a decade ago indi-
cated that N. ceranae is not a new emerging pathogen for
European honey bees and in fact had transferred from its
original host to Apis mellifera earlier than previously
Our studies of N. ceranae infection in European honey
bees in the U.S. confirm and extend the previous observa-
tions by Fries et al. (2006), Higes et al. (2006, 2007) and
Huang et al. (2007) that infection of N. ceranae was not
restricted to its original host. Detection of a pathogen in
bees stored over a long period of time suggests that N. cer-
anae has adapted to and established an infection in Euro-
pean honey bees for quite a while and also demonstrates
the power of PCR technique in handling historical samples
for pathogen infections. However, the discovery of N. cer-
anae in European honey bees raises several questions. First,
assuming A. cerana was the original host of N. ceranae,
when was the exact time that N. ceranae expanded its host
range from A. cerana to A. mellifera? Which transmission
pathway(s) provided opportunities for N. ceranae to infect
new host populations? And while host specificity is a char-
acteristic of microsporidia and the host range of most
microsporidia is relatively narrow (Solter and Becnel,
2003; Solter et al., 2005), how did N. ceranae overcome
the species barrier to expand its host range and establish
infection in a new host? What pathogenic traits of N. cer-
anae provide N. ceranae with the ability to take over the
N. apis and to become a predominant infection in A. melli-
fera in the U.S. populations? In addition, when did this dis-
placement occur, or for how long has Nosema disease in the
U.S. been misclassified as arising solely from N. apis.
Indeed, is it possible that the parasite identified genetically
as N. ceranae (Fries et al., 1996) is in fact the historical
source of this disease in the U.S. The data presented in this
study indicate a strong need for epidemiological and path-
ogenetic studies to identify the conditions that results in
widespread infection of N. ceranae in European honey
Y. Chen et al. / Journal of Invertebrate Pathology 97 (2008) 186–188
Mention of trade names or commercial products in this
article is solely for the purpose of providing specific infor-
mation and does not imply recommendation or endorse-
ment by the U.S. Department of Agriculture.
The authors express their sincere gratitude to Dr. Ste-
phen F. Pernal, from Agriculture Agri-Food Canada for
providing us the N. apis and N. ceranae infected bee sam-
ples, to Michele Hamilton, Nathan Rice, and Andrew
Ulsamer for excellent technical assistance. The work was
supported in part by the 2006 California State Bee-keepers’
Association (CSBA) research fund.
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Fig. 1. PCR amplification of representative bee samples collected from 1995 to 2007 in the United States. For x-axis, No. 1 indicates that DNA was
amplified with generic primers, Nosema F/R; No. 2 and 3 indicates DNA was amplified with specific primers, N. apis F/R and N. ceranae F/R,
respectively. The primer pairs, Nosema F/R, N. apis F/R, and N. ceranae F/R generated PCR fragments of 208, 401, and 250 bp, respectively. DNA that
was extracted from bees collected from Canada and identified to be positive for both N. ceranae and N. apis was used as a positive control (P.C.). Water
was used as a negative control (N.C.). The PCR amplification bands are seen in samples amplified both with primer pairs Nosema F/F and N. ceranae F/R
but in samples amplified with primer pair N. apis F/R.
Y. Chen et al. / Journal of Invertebrate Pathology 97 (2008) 186–188