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JHR 49: 43-50 (20 I 6) JOURNAL OF A peer-reviewed open-access Journal
doi: 10.3897/JHR.49.706 | SHORT COMMUNICATION ME Hymenopter a

http://jhr.pensoft.net The inerrational Society of ymenopterists, RESEARCH

Simultaneous detection of Nosema spp., Ascosphaera
apis and Paenibacillus larvae in honey bee products

Lubiane Guimaraes-Cestaro', José Eduardo Serrao’, Dejair Message’,
Marta Fonseca Martins*, Erica Weinstein Teixeira®

| Doctoral student in entomology, Federal University of Vigosa (UFV), Av. Peter Henry Rolfs, s/n, 36.571-000,
Vicosa, MG, Brazil 2 Department of General Biology (DBG), Federal University of Vigosa (UFV), Av. Peter
Henry Rolfs, s/n, 36.571-000, Vicosa, MG, Brazil 3 Department of Animal Sciences (DCAn), Federal Rural
University of the Semiarid (UFERSA), Km 47-BR110, Mossoré, RN, Brazil 4 Molecular Genetics Laboratory,
Embrapa Dairy Cattle, Rua Eugénio do Nascimento, 610, 36.038-330, Juiz de Fora, MG, Brazil$ Honey bee
Health Laboratory (LASA), Séo Paulo State Agribusiness Technology Agency (APTA —SAA), Caixa Postal 07,
12.400-970, Pindamonhangaba, Sao Paulo, Brazil

Corresponding author: Lubiane Guimardes-Cestaro (lubi.guimaraes@gmail.com)

Academic editor: Jack Neff | Received 2 November 2015 | Accepted 31 March 2016 | Published 28 April 2016
http:/zoobank. org/77B1 DBF 1-463D-4875-9943-390EA3 13664F

Citation: Guimaraes-Cestaro L, Serrao JE, Message D, Martins MF, Teixeira EW (2016) Simultaneous detection of
Nosema spp., Ascosphaera apis and Paenibacillus larvae in honey bee products. Journal of Hymenoptera Research 49:
43-50. doi: 10.3897/JHR.49.7061

Abstract

Honey bees are responsible for pollinating many native and cultivated plant species. These insects can be
affected by many pathogens, including fungi and bacteria, both of which can form spores that are easily
dispersed within the colony by means of the stored products, among other routes. The objective of this
study was to develop a method to detect spores of the honey bee pathogens Nosema apis, Nosema ceranae,
Ascosphaera apis and Paenibacillus larvae in samples of honey, bee pollen and royal jelly. The method was
standardized for each product individually, and then analyzed by monoplex and multiplex PCR, which
showed the same detection thresholds: 1.25 spores/mL of honey for NV. ceranae; 7.5 spores/mL of honey
for A. apis; and 0.4 spore/mL of honey for P /arvae, respectively. The standardized technique was effective
and rapid for the detection of these pathogens in bee products and can be used for the establishment of
official methods of sanitary control of bee products, considering the growing national and international

trade of these products and the movement or migration of colonies between regions.
Keywords

Apis mellifera, Bee pathogens, Honey, Pollen, Royal Jelly, Detection of spores

Copyright Lubiane Guimardes-Cestaro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

44 Lubiane Guimaréaes-Cestaro et al. / Journal of Hymenoptera Research 49; 43—50 (2016)

Introduction

The honey bee, Apis mellifera, is essential to global agriculture by pollinating a wide range
of food crops, besides supplying economically important products (Evans and Schwarz
2011). These insects can be affected by various pathogens (Bailey and Ball 1991), in-
cluding the bacterium Paenibacillus larvae and the fungi Ascosphaera apis, Nosema apis
and Nosema ceranae. ‘These fungi stand out for their ease of transmission in the form of
spores, which can remain viable in the environment for many years, transmitting diseases
between colonies and among their individuals (Shimanuki and Knox 1994).

Within the colony, products like honey, pollen and royal jelly are susceptible
to contamination by these pathogens, mainly through storage in honeycomb and
trophallaxis (OIE 2014). In addition, some negligent hygienic practices by beekeep-
ers, such as reuse of apiculture materials (Van der Zee 2010), exchange of combs con-
taining the remains of diseased broods (OIE 2014), and reuse of contaminated wax
(Malone and Gatehouse 1998; OIE 2014), among other practices (Higes et al. 2008;
Aronstein and Murray 2010), are indicated as important routes for dissemination of
these pathological agents (Hale and Menapace 1980; Higes et al. 2008; Giersch et
al. 2009). For this reason, research has been carried out to develop methods to detect
pathogens in bee products, mainly involving the use of polymerase chain reaction
(PCR) (de Graaf et al. 2013).

Multiplex PCR is used if more than one pathogen is amplified simultaneously in
a single reaction, resulting in considerable savings of both time and effort (Sigh et al.
2000), besides reducing costs of reagents and allowing fast analysis of a large number
of samples. Thus, molecular biology techniques such as multiplex PCR are important
tools for the detection of pathogens.

The aim of this study was to develop a method to detect spores of NV. apis, N. ceranae,
A. apis and P. larvae in honey, bee pollen and royal jelly samples.

Materials and methods

Solutions containing spores of Nosema ceranae and Ascosphaera apis were prepared from
naturally contaminated bees according the protocols described by Teixeira and Mes-
sage (2010) and Cantwell (1970). For the Paenibacillus larvae spore suspension, we
were used a solution of ATCC 9545, supplied by the National Agriculture Labora-
tory - Ministry of Agriculture. The solutions were inoculated in different quantities of
sterile bee products, previously submitted to cobalt irradiation, receiving an ion beam
dose of approximately 960 Gy/10 cm: honey (5 mL, 10 mL and 20 mL), pollen (0.5
g, 1 g,5 gand 10 g) and royal jelly (0.5 g, 1 g, 5 g and 10 g), which were submitted to
different analytic processes (homogenization, filtration and centrifugation) to test the
spore concentrations. The presence of pathogens was confirmed by PCR and sequenc-
ing before the tests.

Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae... 45

For detection of the pathogens, the samples were weighed (10 g for pollen, 5 g for
royal jelly and 20 mL for honey) in sterile tubes and diluted in 40 mL of sterile dis-
tilled water. The samples of pollen were diluted in 45 mL of sterile distilled water. The
samples were vigorously homogenized and centrifuged at 12,500 x g for 40 min. The
supernatant was discarded and the pellet was resuspended in 1 mL of sterile distilled
water, with subsequent homogenization and centrifugation at 10,000 x g for 20 min.
The supernatant was again discarded and the pellet was submitted to DNA extraction
using the Qiagen DNeasy® Plant Mini Kit, following the manufacturer’s instructions.
In the case of bee pollen, after homogenization the samples were filtered with What-
man | filter paper using a vacuum pump and then the same procedure was followed as
for the samples of honey and royal jelly.

Monoplex and multiplex PCR were carried out following Puker (2011) to con-
firm the detection of each pathogen in the products analyzed. These reactions were
performed with a volume of 20 wL, composed of 10 uL of GoTaq® Green (Promega),
8 uL of DNAse free water, 1 wL of sample DNA and 1 pL of each primer (10 1M)
(Piccini et al. (2002), Murray et al. (2005) and Martin-Hernandez et al. (2007) for
P. larvae, A. apis and N. apis! N. ceranae, respectively). All analyses were carried out
with positive and negative controls. Spores of NV. ceranae removed from forage bees
naturally infected with the microsporidian were used as positive control. The positive
controls of NV. apis was sent by the Bee Research Laboratory — USDA, considering the
low prevalence of the pathogen in Brazil (0.39%) (Teixeira et al. 2013), causing it
often not to be detected (Santos et al. 2014). As positive control of P. larvae, spores of
ATCC 9545 supplied by Lanagro were used.

Decreasing spore concentrations were tested (2500, 250, 100, 50, 25 spores of
Nosema ceranae; 3000, 300, 150, 75 spores of Ascosphaera apis; 1000, 100, 10, 8, 4,
1 spores of Paenibacillus larvae), until reaching the minimum detectable number, in
order to identify the technique’s sensitivity. All the analyses were performed at the
Honey bee Health Laboratory of Sao Paulo State Agribusiness Technology Agency
(LASA/APTA), located in Pindamonhangaba, Sao Paulo.

Results

Multiplex PCR allowed the simultaneous detection of NV. ceranae, A. apis and P larvae in
different bee products, and the amplified product for each pathogen presented the expect-
ed fragment lengths (218 bp, 485 bp and 700 bp, respectively). Threshold values were the
same for the monoplex and multiplex reactions (Figure 1). In samples of honey, we found
1.25 spores/mL of honey for Nosema ceranae (218 bp), 7.5 spores/mL for Ascosphaera apis
(485 bp) and 0.4 spore/mL for Paenibacillus larvae (700 bp). For bee pollen we found 2.5
spores/g of pollen for NV. ceranae (218 bp), 15 spores/g for A. apis (485 bp) and 0.8 spore/g
for P larvae (700 bp). For samples of royal jelly we found 5 spores/g of royal jelly for NV.
ceranae (218 bp), 30 spores/g for A. apis (485 bp) and 1.6 spores/g for P larvae (700 bp).

46 Lubiane Guimardaes-Cestaro et al. / Journal of Hymenoptera Research 49: 43-50 (2016)

PF lever (70 bp) ~—>

—

Figure |. Agarose gel (2%) stained with SYBR® Safe of the monoplex and multiplex PCR products
referring to the detection thresholds of the pathogens in honey bee products. M: 100 bp marker. I 2.5
spores/g of pollen for Nosema ceranae 2 15 spores/g of pollen for Ascosphaera apis 3 0.8 spore/g of pollen
for Paenibacillus larvae 4 multiplex PCR for N. ceranae (2.5 spores/g of pollen), A. apis (15 spores/g of
pollen) and P. larvae (0.8 spore/g of pollen) 5 multiplex positive controls for N. ceranae (218 bp), N. apis
(321 bp), A. apis (485 bp) and P. larvae (700 bp) 6 negative control. M: 100 bp marker 7 1.25 spores/
mL of honey for NV. ceranae 8 7.5 spores/mL of honey for A. apis 9 0.4 spore/mL of honey for P. larvae
10 multiplex PCR for NV. ceranae (1.25 spores/mL of honey), A. apis (7.5 spores/mL of honey) and P. /ar-
vae (0.4 spore/mL of honey) II multiplex positive controls for NV. ceranae (218 bp), N. apis (321 bp), A.
apis (485 bp) and P. larvae (700 bp) 12 negative control. M: 100 bp marker 13 5 spores/g of royal jelly for
N. ceranae \4 30 spores/g of royal jelly for A. apis 15 1.6 spores/g of royal jelly for P. larvae 16 multiplex
PCR for WN. ceranae (5 spores/g of royal jelly), A. apis (30 spores/g of royal jelly) and P. darvae (1.6 spores/g
of royal jelly) 17 multiplex positive controls for N. ceranae (218 bp), N. apis (321 bp), A. apis (485 bp)
and P. larvae (700 bp) 18 negative control.

Because JV. apis occurs in very low prevalence (Teixeira et al., 2013), and is dif-
ficult to detect in naturally infected samples, spores of this species were not used in
the standardization. We believe the detection threshold would be the same as that
obtained for NV. ceranae (Martin-Hernandez et al. 2007), where the amplification when
compared to the positive control worked perfectly.

Discussion

Detection of Nosema apis, N. ceranae, Ascosphaera apis and Paenibacillus larvae in sam-
ples of bee products has been reported (Cox-Foster et al. 2007, Higes et al. 2008,
Giersch et al. 2009), but few studies have reported the detection limits.

Puker (2011) centrifuged samples of honey at 10,000 x g for 40 min and reported
detection limits of 10 and 100 spores/mL of honey for Ascosphaera apis in monoplex
and multiplex reactions, respectively, 100 spores/mL of honey for Nosema ceranae, and
10 spores/mL of honey in monoplex and multiplex reactions for P. /arvae. Our results
showed lower limits of detection (1.25 spores / mL honey for NV. ceranae, 7.5 spores /
mL for A. apis mel and 0.4 spores / mL honey for Paenibacillus larvae, which can be
attributed to the increase in the centrifugation speed and time.

Some studies have presented detection limits that corroborate this idea. Piccini et
al. (2002) obtained a detection limit of 32 spores/mL of honey, analyzing 10 mL of
honey and centrifuging the samples at 6,000 x g for 20 min. D’Alessandro et al. (2006)

Simultaneous detection of Nosema spp., Ascosphaera apis and Paenibacillus larvae... 47

also analyzed honey and found a detection limits of 20 spores/reaction, using 20 mL of
honey and centrifuging the samples at 6,000 x g for 45 min. In these studies, the limits
of detection decreased when the spin time was increased.

Although the technique presented here is similar to those employed by other au-
thors, our tests showed that the samples centrifuged below 12,000 x g did not undergo
sedimentation, and consequently a considerable number of spores were still present in
the supernatant.

To assure that no spores remained in the supernatant, besides performing a centrif-
ugation at 12,000 x g for 40 min, we also submitted the resuspended pellet obtained
to new centrifuging at 10,000 x g for 20 min, for subsequent extraction of DNA. ‘This
factor is likely responsible for the technique’s sensitivity.

For detection of the bacterium Paenibacillus larvae, various methods have been de-
scribed, mainly using growth in culture medium and PCR (de Graaf et al. 2013; OIE
2014). The Manual of OIE (2014) describes detection of spores in suspension in samples of
pollen and royal jelly, employing centrifuging at 6,000 x g during 30 min. This centrifuge
force can be considered low compared to that used here, which may have left spores in the
supernatant, thus hindering efficient detection of the pathogens contained in the samples.

According to the OIE (2014), for the analysis of Paenibacillus larvae in pollen, 1
g of the product in 10 mL of sterile distilled water or PBS should be filtered through
Whatman 1 filter paper for subsequent microbiological analysis. We also suggest this
filtration of pollen samples, because without this step the samples still contained a large
quantity of material after centrifuging, which can have a negative effect on extraction
of the genetic material and also on the PCR reactions.

Microbiological techniques have also been used to detect Paenibacillus larvae, but
it is important to consider the time for analysis and efficacy in detecting this bacterium.
According to the official technique specified in Brazil (Brasil 2003), it is necessary to
wait five days for colony growth in PLA culture medium, plus over three days for con-
firmatory tests. In other suggested microbiological techniques, it is necessary to incubate
the bacteria in culture medium for 6, 7 and 8 days (de Graaf et al. 2013; OIE 2014). The
time frame thus varies from 5 to 11 days, placing a significant burden on beekeepers.

The protocol developed showed important results when used to analyze samples
of bee products marketed in Sao Paulo state (in preparation), supporting the idea that
PCR is a fast and reliable technique to diagnose pathogen infections (Martin-Hernan-
dez et al. 2007; de Graaf et al. 2013).

The multiplex PCR method offers a significant cost-saving advantage, especially
when large numbers of samples are analyzed (Sguazza et al. 2013). Furthermore, mul-
tiplex PCR is able to detect multiple target DNA sequences in a single reaction, with
the simultaneous identification of two or more kinds of pathogens, simplifying the
workflow and processing time, which are significantly reduced (Bilgic et al. 2013),
among other advantages (Edwards and Gibbs 1994).

The protocol presented here is useful to detect simultaneously Nosema apis, Nose-
ma ceranae, Ascosphaera apis and Paenibacillus larvae in samples of honey, pollen and
royal jelly and can support definition of official methods for surveillance actions.

48 Lubiane Guimardaes-Cestaro et al. / Journal of Hymenoptera Research 49; 43-50 (2016)

Acknowledgements

This work was supported by the Sao Paulo State Research Foundation (FAPESP, EWT
2012/18802-3). We thank the Office to Improve University Personnel (CAPES) for
the scholarship funding, the Sao Paulo State Agribusiness Technology Agency (APTA,
SAA-SP) for the institutional support, and Maria Luisa T. M. F. Alves for her help
with the analyses. MFM is a research fellow of the National Council for Scientific and
Technological Development (CNPq). JES is a research fellow of the CNPq and Minas
Gerais Research Foundation (FAPEMIG).

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