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Advances in the apiary control of the honeybee American Foulbrood with Cinnamon(Cinnamomum zeylanicum) essential oil

Authors:
  • Instituto de Investigaciones en Producción Sanidad y Ambiente (IIPROSAM) UNMDP - CONICET
  • Instituto de Investigación en Producción Sanidad y Ambiente (IIPROSAM) Universidad Nacional de Mar del Plata

Abstract

The activity of Cinnamomum zeylanicum Nees essential oil (cinnamon oil) against Paenibacillus larvae (Withe) was evaluated in the laboratory and in a field experiment in order to improve the biological control of the Apis mellifera L. disease American foul-brood (AFB). The MICs (Minimal Inhibitory Concentration) against P. larvae were determined by the tube dilution method. Bee lethality was estimated using cages with approximately 374 bees, fed with the essential oil at different concentrations diluted in ethanolic syrup. The security index was calculated as LD50 of A. mellifera/MIC of P. larvae strain at 24, 48 and 72 h in both tests. The apiary trial, using three groups of five hives each, was carried out in April-May 2006 in an experimental apiary of J. J. Nágera coastal station (Mar del Plata, Argentine). The first group was treated with cinnamon oil (two doses of 1000 µg/ml per hive), the second with oxytracycline-HCl (three doses of 0.4 g per hive) and the third was left untreated as a control. All of the treatments were performed at 7-day intervals. The evaluation of treatment efficacy was made by counting the number of infected brood cells in both sides of a central comb, using a brood surface of 360 cm 2 (18 x 20 cm). For the Mar del Plata AFB strain the C. zeylanicum essential oil and antibiotic MICs were 50 µg/ml and 3.125 µg/ml, respectively. Regarding the systemic administra-tion method, bees LD50 at 24 h was 456.07 µg of essential oil of cinnamon/bee. LD50 estimated at 48 h showed a slight decrease with respect to that recorded at 24 h. Security index was 9.1214 (ml/bee) for 24 h. In relation with field trials, after 24 and 31 days from the beginning of treatments the cinnamon oil-treated hives showed a lesser incidence of infected larvae (7.89% and 52.42%) than the control group highlighting a clear efficient control. No significant differences between the two treated groups were re-corded. These results represent a further proof of the potential of cinnamon oil to control American Foulbrood with only minor toxicological risks to bees. Furthermore, the use of cinnamon oil avoids problems with antibiotic residues in honey. This is impor-tant in marketing honey in the EU where antibiotics in honey are generally forbidden by law.
Bulletin of Insectology 62 (1): 93-97, 2009
ISSN 1721-8861
Advances in the apiary control of the
honeybee American Foulbrood with Cinnamon
(Cinnamomum zeylanicum) essential oil
Liesel Brenda GENDE
1,2,3
, Matías Daniel MAGGI
1,3
, Natalia DAMIANI
1,3
, Rosalia FRITZ
2
, Martin Javier
EGUARAS
1,3
, Ignazio FLORIS
4
1
Departamento de Biología, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Argentina
2
Departamento de Química, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Argentina
3
CONICET, Universidad Nacional de Mar del Plata, Argentina
4
Dipartimento Protezione delle Piante, Sezione Entomologia agraria, Università degli Studi di Sassari, Italy
Abstract
The activity of Cinnamomum zeylanicum Nees essential oil (cinnamon oil) against Paenibacillus larvae (Withe) was evaluated in
the laboratory and in a field experiment in order to improve the biological control of the Apis mellifera L. disease American foul-
brood (AFB). The MICs (Minimal Inhibitory Concentration) against P. larvae were determined by the tube dilution method. Bee
lethality was estimated using cages with approximately 374 bees, fed with the essential oil at different concentrations diluted in
ethanolic syrup. The security index was calculated as LD50 of A. mellifera/MIC of P. larvae strain at 24, 48 and 72 h in both
tests. The apiary trial, using three groups of five hives each, was carried out in April-May 2006 in an experimental apiary of J. J.
Nágera coastal station (Mar del Plata, Argentine). The first group was treated with cinnamon oil (two doses of 1000 µg/ml per
hive), the second with oxytracycline-HCl (three doses of 0.4 g per hive) and the third was left untreated as a control. All of the
treatments were performed at 7-day intervals. The evaluation of treatment efficacy was made by counting the number of infected
brood cells in both sides of a central comb, using a brood surface of 360 cm
2
(18 x 20 cm). For the Mar del Plata AFB strain the
C. zeylanicum essential oil and antibiotic MICs were 50 µg/ml and 3.125 µg/ml, respectively. Regarding the systemic administra-
tion method, bees LD50 at 24 h was 456.07 µg of essential oil of cinnamon/bee. LD50 estimated at 48 h showed a slight decrease
with respect to that recorded at 24 h. Security index was 9.1214 (ml/bee) for 24 h. In relation with field trials, after 24 and 31 days
from the beginning of treatments the cinnamon oil-treated hives showed a lesser incidence of infected larvae (7.89% and 52.42%)
than the control group highlighting a clear efficient control. No significant differences between the two treated groups were re-
corded. These results represent a further proof of the potential of cinnamon oil to control American Foulbrood with only minor
toxicological risks to bees. Furthermore, the use of cinnamon oil avoids problems with antibiotic residues in honey. This is impor-
tant in marketing honey in the EU where antibiotics in honey are generally forbidden by law.
Key words: Cinnamon oil, oxytetracycline, antimicrobial activity, American Foulbrood control, apiary trial.
Introduction
The American Foulbrood (AFB) is considered the most
serious bacterial disease of honeybees (Shimanuki and
Knox, 1997). It is caused by the spore-forming bacte-
rium Paenibacillus larvae (Withe) (Genersch et al.,
2006) that affects the larval stage of honeybees (Apis
mellifera L.), with a rapid widespread and destructive
effects on the colony. The spores are extremely infec-
tious and resistant to physical and chemical agents
(Haseman, 1961). A dead larva may contain billions of
spores (Hansen and Brødsgaard, 1999). The use of anti-
biotics in hives is illegal in some EU countries because
up to now there are no formulations that have obtained
the necessary Ministerial registration for manufacture,
transport, and sale. Moreover, there are no MRLs
(Maximum Residue Limits) of tetracyclines and sul-
phonamides established for honey according the Euro-
pean Community regulations (Mutinelli, 2003). Only in
exceptional cases, the veterinary authority can authorize
in the prophylaxis of this disease the employment of an-
tibiotic formulations registered for other veterinary use
alone or in association with manipulative treatments,
such as the “shaking” method, consisting in transferring
bees from diseased colonies onto new combs into empty
box. However, affected honeybees colonies often have
to be destroyed and the equipment adequately decon-
taminated (Bailey and Ball, 1991). In other countries as
in the USA, Canada and Argentina, preventive treat-
ments with antibiotics are allowed, being considered a
routine procedure to prevent outbreaks of AFB (Lind-
strom, 2006). Consequently, various strains of P. larvae
showing resistance to antibiotics, such as oxytetracy-
cline-HCl (OTC), have been discovered in Argentina
(Alippi, 2000) as well as in many United States areas
(Miyagi et al., 2000). Moreover, the extensive use of
antibiotics leads to an accumulation of residues in bee-
hive products (especially in honey), decreasing their
quality and making their marketing more difficult (Fu-
selli et al., 2005).
Because of legal and biological issues associated with
antibiotic use in hives, we have been examining natural
antimicrobial products for AFB management. Based on
previous experiences (Gende et al., 2008b), we chose to
study the antimicrobial activity of cinnamon oil against
a specific Argentinean strain of P. larvae and the oils
toxicity to A. mellifera using both laboratory and field
tests.
94
Materials and methods
Essential oil
Cinnamon essential oil, was furnished by Cruciani
Company (Rome, Italy) and derived from the bark of
Cinnamomum zeylanicum Nees, the essential oil/product
was the same previously employed and analysed by Flo-
ris et al. (1996).
Antimicrobial assay
A bacterial strain of P. larvae was isolated from hon-
eybee brood combs of colonies with clinical symptoms
of AFB; these were collected in hives located in Mar del
Plata (38°0'S-57°33'W), Buenos Aires province. The
Argentinean strain of P. larvae was previously identi-
fied in laboratory by biochemical assays (Alippi, 1992)
and tests based on PCR and restriction fragment analy-
sis of the 16S rRNA genes (rDNA), using the primers
PL 5 (5’-CGAGCGGACCTTGTGTTTCC-3’) and PL 4
(5’-TCAGTTATAGGCCAGAAAGC-3), which am-
plify a fragment of the P. larvae 16S rRNA gene (Pic-
cini et al., 2002; D’Alessandro et al., 2006). The pure
strain was maintained on MYPGP agar with 15% v/v
glycerol until used. Vegetative cells of P. larvae were
grown on MYPGP agar for 48 h at 35 ± 0.5 °C, and then
suspended in double distilled autoclaved water. To
measure antimicrobial activity with serial dilution
method, microbial biomass concentration was adjusted
to 0.5 of MacFarland scale (Scott, 2006). The minimal
inhibitory concentration (MIC) of cinnamon essential
oils and antibiotic (oxytetracycline-HCl) separately, was
directly assessed by turbidity observation. Stock solu-
tions of each antimicrobial agent in sterile water were
made; essential oil of cinnamon was emulsified with 8%
v/v propylene glycol (1-2 propanediol). One milliliter of
each stock solution was added to MYT broth (Gende et
al., 2008a) and serially diluted (final range 12.5-2000
µg/ml from essential oil and 0.000315-100 µg/ml for
antibiotic). Microbial biomass suspension was then
added to each serial dilution tube with agitation, at room
temperature, using a Vortex dispersing tool (Fbr
®
by
Decalab Srl). All sample tubes (as well as positive and
negative controls) were incubated at 35 ± 0.5 °C for 48
h in order to determine MIC values under microaerobic
conditions (5-10% of CO
2
). All MIC tests were per-
formed by triplicate for microbial agent and strain.
Bees lethality test
To evaluate the oil effect on A. mellifera, an average
of 374 ± 45 adult worker bees taken from the nest were
placed in each cage (16 x 12 x 6 cm) with nylon mesh
on the walls for air circulation. After being starved for 4
hours, to allow bees to feed during the experiment, a
cylindrical feeder (8 x 3.5 cm) were placed inside each
cage (Maggi et al., 2009) with 10 ml of sugar-water
syrup (2:1) in 70% ethanolic solution containing 2000,
4000, 8000, 16000 µg/ml of cinnamon oil concentra-
tions, respectively. Bees in cages fed with sugar syrup
in 70% ethanolic solution without oil and other treated
with dimethoate were also included as controls. Each
treatment was replicated five times. After 24 h, the
cages received only 2:1 syrup, depending on demand of
bees. All cages were maintained at room temperature
(22 °C and 65% RH) and a synthetic queen pheromone
was also introduced in them. Bee mortality was re-
corded at 24, 48 and 72 h. Once the experiment fin-
ished, the bees were killed by immersing the cages in a
70% alcoholic solution container. The final number of
bees was recorded to calculate the proportion of dead
bees attributable to each time (24, 48 and 72 h), and sta-
tistical analysis was made by linear regression method.
The Security Index was determined as LD50 of A.
mellifera/ MIC of P. larvae, modifying the expression
given by Lindberg et al. (2000).
Apiary trial
The field trial was carried out from April to May 2006
in the Arthropods Laboratory at the Universidad Na-
cional de Mar del Plata (Argentine) and in an experi-
mental apiary of J. J. Nágera coastal station placed on
route 11 Km 32 (38°10'06"S, 57°38'10"W).
Twenty days before treatments, 15 nuclei of homoge-
neously strong-bees were prepared; each colony consists
of 5-6 combs of bees. Capped and open broods as well as
food reserves were included in same proportion. The
colonies from which these combs were extracted had not
been treated with antibiotics during the 24 months prior
the experiment. The young queens were marked to be
easily recognized. Hives were divided into three groups
of five. All the hives were artificially infected with the
same number of scales of pre-pupae or pupae affected by
AFB. Inoculation was made with brood comb sections (5
x 5 cm) collected from colonies showing AFB clinical
symptoms. Each infected comb section had 45 ± 5 scales
and they were introduced into nuclei in central position
of a nest central comb. After 3 days, the piece of incor-
porated brood comb was modified in its entirety by the
bees and converted with new wax, giving up the comb of
experimentation with normal and similar characteristics
to the remaining breeding ones. The Argentinean strain
of P. larvae used in the artificial inoculation was the
same that had been analyzed previously.
The first group of hives (O) was treated with oxytetra-
cycline-HCl and received three weekly doses, each of
0.4 g antibiotic mixed with an equal quantity of pow-
dered sugar. The second group (Ci) received two
weekly treatments of 250 ml syrup (solution 2:1 of
sugar/water) added with cinnamon oil at a concentration
of 1000 µg/ml. In this case, it was also necessary to use
an emulsifier of 70% ethanol in the syrup. The third
group (Co) was left as a control and received only etha-
nolic syrup (solution 2:1 of sugar/water with ethanol).
The evaluation of AFB disease incidence following
the treatments was made by counting the number of in-
fected brood cells in both sides of a central comb por-
tion of 360 cm
2
(18 x 20 cm). For each hive, the number
of cells with infected larvae and/or scales and the num-
ber of cells with healthy larvae were weekly counted
until the 31
st
day after the experimental infection.
Statistical analysis
Experimental data were analyzed by analysis of vari-
ance (ANOVA) after arcsine ¥ y/100) transformation, in
the case of percentages, to reduce the heterogeneity of
95
Table 1. Medium lethal dose (LD50) (µg of cinnamon essential oil/bee) at different exposure-time intervals.
Hours Regression equation R
2
LD50 (µg essential oil/bee)
24 y = 0.0031 x í2.82144 0.5419 456.07
48 y = 0.00316 x í1.01723 0.5327 432.13
72 y = 0.00316 x í0.44988 0.5319 427.32
Table 2. Security index values (SI), ratio between medium lethal concentration (µg of cinnamon essential oil/bee)
and minimal inhibitory concentration (MIC, µg/ ml) of essential oil against P. larvae.
Hours LD50 (µg essential oil/bee) MIC (µg/ml) SI (ml/bee)
24 456.07 50 9.1214
48 432.13 50 8.6426
72 427.32 50 8.5464
Table 3. Mean values (percentage ± s.d.) of infected larvae at different days from experimental infection.
Days Control Essential oil Antibiotic F-value
17 0.59 ±0.30 a 0.14 ± 0.13 b 0.23 ± 0.31 b 4.54 (P = 0.0341)
24 13.08 ± 5.23 a 5.19 ± 2.95 b 4.75 ± 3.41 b 6.82 (P = 0.0105)
31 83.91 ± 13.39 a 31.49 ± 5.84 b 19.06 ± 6.3 b 29.39 (P = 0.0000)
Means in each line followed by different letters are significantly different (LSD test, P < 0.05).
the variance. When the F-tests were significant, means
were separated applying the least significant difference
(LSD) test (P < 0.05) (Statgraphics Plus 2001). Tables
show non-transformed values.
Results
Antimicrobial assay
Against P. larvae strain used in these experiments,
MIC values were 50 µg/ml and 3.125 µg/ml respectively
for the essential oil of C. zeylanicum and for oxytetracy-
cline-HCl.
Bees lethality test
Estimated A. mellifera LD50s were 456.07 µg/bee,
432.13 µg/bee and 427.32 µg/bee at 24 h, 48 h and 72 h,
respectively (table 1). Referring to the ICBB (1985), the
theoretical LD50 values for the bees of specific active
ingredient, like dimethoate, range between 0.1-0.3
µg/bee; this level corresponds to a highly toxic com-
pound (Gough et al., 1994). Therefore, the highest
LD50 value for the C. zeylanicum essential oil, would
correspond to a "virtually non-toxic" product, according
to the ICBB (1985).
As an adaptation of the methodology used by
Lindberg et al. (2000) the security index was employed
to estimate a safe factor of the colony (table 2). This pa-
rameter diminished after 24 h, because LD50 values de-
creased over time, while MIC values remained stable.
This trend shows that when oil exposure-time of bees
increases, toxicity is expected to grow.
Apiary trial
After the artificial infection of bee colonies, the ex-
perimental model allowed to follow the development of
the disease in all beehives. From the 3
rd
to the 5
th
day
after the scales inoculation, the bees cleaned the in-
fected cells and the queen laid eggs inside. After the 10
th
day, infected larvae were not detected during inspec-
tions. From the beginning of the treatments to the 17
th
day, colonies treated with essential oil showed an aver-
age of 0.14% infected larvae, which is similar to levels
recorded in colonies treated with antibiotics, but lower
than untreated ones. After the 24
th
day, colonies treated
with essential oil showed a higher number of infected
larvae than those treated with antibiotics, but even lar-
ger numbers of infected larvae were recorded in the
control hives. Significant differences (P < 0.05) among
treated and control groups were verified (table 3). From
the beginning of the treatments to the 24
th
and 31
st
day,
the essential oil-treated hives group showed a reduction
in infected larvae percentages of 7.89% and 52.42%,
respectively, in comparison with the untreated ones. By
contrast, these values increased of 0.44% and 12.43%,
respectively, when compared to the antibiotic treated
group. However, no statistical differences were recorded
between the two treated groups (table 3).
Discussion and conclusions
MIC values of cinnamon essential oil obtained from
Mar del Plata AFB strain were similar to those obtained
against others P. larvae strains isolated from infected
honeybee brood combs of Buenos Aires province
(Gende et al., 2008b). In relation with minimal inhibi-
tory concentration of the antibiotic, the Mar del Plata
strain was susceptible by introducing a value of MIC
less than 5 µg/ml (Alippi, 1996).
When the essential oil was administered in sugar syrup
by systemic way, regression equations presented in table
96
1 shows that the mortality in bees fed with 1000 µg/ml
syrup did not exceed 10.0% on any day. Although the
oral toxicity of essential oils has been already studied on
the bees (Ebert et al., 2007), following the preliminary
test carried out by Carta and Floris (1989), to our knowl-
edge this is the first report regarding the use of cinnamon
essential oil by oral administration in vitro assays.
In the experimental apiary control beehives showed
higher levels of infected cells than treated ones. There-
fore, both tested products, the antibiotic and the cinna-
mon oil, were effective in the control of AFB. However,
as already known, the use of antibiotics compared to the
essential oil can generate toxicological and biological
risks due to an accumulation of residues in beehive
products which decreases their quality and can also in-
duce resistance to P. larvae strains or other bee patho-
gens (Miyagi et al. 2000; Evans, 2003; Thompson et al.,
2005; Martel et al., 2006). Although we can exclude
that the Argentinean strain of P. larvae tested is par-
tially resistant to antibiotics (Alippi, 1996), the analo-
gous disease development in the treated groups (O and
Co), proved that cinnamon oil may represent an impor-
tant alternative natural product for AFB control in api-
ary. Its efficacy was statistically comparable to the
tested antibiotic. The results of our experiments support
other research findings (Carta and Floris, 1989; Carpana
et al. 1996; Floris et al., 1996; Floris, 2001). Moreover,
several recent studies proved the use of cinnamon as a
stimulus for honey bee learning (Abramson et al., 2006,
2008) or its use against other important mites and dis-
ease vectors (Abramson et al., 2007). In addition, the
apparently lower ability of other natural essence-based
products to keep down bacterial infestation levels (Albo
et al., 2003), encourages the employment of cinnamon
oil in the hives. Nevertheless, in our case the experi-
mental infection was based on an initial inoculum corre-
sponding to a very high dosage of bacterial spores in
comparison with a natural infection. In fact, a dry scale
formed from a diseased larva affected by AFB usually
contains approximately 2 x 10
9
spores/bee (Shimanuki
and Knox, 1997); in our tests an average number of 45 ±
5 scales corresponding to about 9 x 10
10
spores/bee,
were introduced in the previously healthy beehives. On
the other hand, it is generally agreed that the use of
naturally infected beehives better mimic the reality and
consequently provide more reliable treatment efficacy
evaluations. Beyond, in order to avoid the possible loss
of honeybee orientation observed by Higes et al. (1997),
we included only two doses of cinnamon essential oil in
two weekly treatments. So that, enhanced efficacy of
cinnamon oil in comparison with antibiotic treatments,
might have been obtained by either using three different
doses or with an experimental infection based on a
lower number of scales. Finally further improvements
might be obtained employing different and more appro-
priate treatment application techniques. For instance,
manipulative treatments, such as the cited “shaking”
method (Bailey and Ball, 1991) aimed at salvaging adult
bees from combs containing diseased brood, before the
administration of cinnamon oil, is supposed to provide
optimal efficacy in preventing AFB. Enhancement of
treatment efficacy might be possible also if this oil is
used in combination with other active ingredients such
as thymol (Fuselli et al., 2006); plant extracts, other es-
sential oils and their principal components (Gende et al.,
2008b; 2008c; 2009) involved in integrated pest man-
agement strategies of the hive.
Everything considered, this study represents a further
progress of a larger research, involving the chemical
composition and the concentration of the main cinna-
mon essential oil compounds, with special regard to the
components inhibiting bacterial growth (Gende et al.,
2008b). All these findings have helped to define rec-
ommended concentrations for apiary application in or-
der to minimize the toxicological risks for bees and to
avoid the honey taste threshold (Bogdanov et al., 1999)
or other undesirable effects, such as the appearance of
resistance strains as a consequence of the indiscriminate
use of antibiotics.
Acknowledgements
The authors would like to thank Claudia Faverin for the
assistance in providing the statistical analysis also Ser-
gio Ruffinengo and Gabriel Sarlo for their help in the
fields experiments and to Giovanni Formato for critical
review and Michael Robeson for English revision. This
work was supported by UNMDP, ANPCyT and CONI-
CET.
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Authors’ addresses: Ignazio F
LORIS (corresponding au-
thor: ifloris@uniss.it), Dipartimento Protezione Piante, Sezio-
ne Entomologia, Università degli Studi di Sassari, via E. De
Nicola, 07100 Sassari, Italy; Liesel Brenda G
ENDE
(lgende@mdp.edu.ar), Matías Daniel M
AGGI (biomaggi@
gmail.com), Natalia D
AMIANI (ndamiani@mdp.edu.ar), Rosa-
lia F
RITZ (rfritz@mdp.edu.ar), Martin Javier EGUARAS
(meguaras@mdp.edu.ar), Universidad Nacional de Mar del
Plata, Funes 3350 (7600), Mar del Plata, Buenos Aires, Argen-
tina.
Received September 4, 2008. Accepted March 15, 2009.
... Bee venom has been also proposed as an antimicrobial agent of control . Meanwhile, cinnamon oil has been successfully proved to control AFB in apiary trials (Gende et al. 2009). ...
... However, field trials should be performed to know the effective dose of substance to apply in natural conditions. As a reference, in the work of Gende et al. (2009), cinnamon (Cinnamomum zeylanicum ) essential oil showed a MIC of 50 ppm on P. larvae , whereas applications of 1000 ppm were needed to reduce the level of AFB in hives. Therefore, it would be expected that doses of around 460 ppm of NP2 are effective under field conditions, much lower values than the LD 50 ones obtained for worker bees (96,000 ppm) and larvae (36,000 ppm). ...
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The aim of this work was to evaluate antimicrobial activity against Paenibacillus larvae and oral toxicity against workers and larvae of Apis mellifera of gallic acid (GA) and two nanohybrids of GA and silica. Also, the physicochemical, structural, and energetic properties of GA and the nanohybrids were determined through structure–activity relationship (SAR). The minimum inhibitory concentration (MIC) against P. larvae was determined. GA showed MIC values between 62.5 and 125 μg/ml, whereas the nanoparticle functionalized through the GA carboxylic moiety (NP2) showed the best antimicrobial activity with a MIC value of 23 μg GA/ml for four of the five isolates used. SAR analysis showed that electronegativity, chemical hardness, and dipolar moment are reliable estimators of the antimicrobial activity. NP2 showed the lowest toxicity against workers and was innocuous for bee larvae. Therefore, the nanohybrid NP2 was the best antibacterial and resulted in non-toxic against workers and larvae of honeybees, becoming a potentially effective and safe agent for the treatment of American Foulbrood disease.
... Debido a la ilegalidad del uso de productos sintéticos, como acaricidas, fungicidas y antibióticos para el tratamiento de diversas afectaciones causadas por ácaros (Varroa destructor, Acarapis woodi), hongos (Nosema apis, Nosema ceranae) y bacterias (Paenibacillus larvae, Mellisococus plutonium, Ascophaera apis), que afectan la salud de las abejas (Apis mellifera), recientemente se han empezado a evaluar aceites esenciales para el control de Paenibacillus larvae y Varroa destructor, por lo que se han utilizado diversos aceites como aceite esencial de ajo, orégano, sábila, manzano, ruda, romero, menta y canela, este último se ha estudiado recientemente para el control de loque americana (Paenibacillus larvae) debido a su actividad antimicrobiana, puesto que se ha demostrado que las colmenas tratadas con aceite de canela presentan una menor incidencia de larvas infectadas con Paenibacillus larvae en comparación con el grupo control (Gende et al., 2009), estos resultados representan una prueba más del potencial del aceite de canela para controlar la Paenibacillus larvae con riesgos toxicológicos menores para las abejas, además que se ha demostrado que las colmenas tratadas con aceite de canela, tiene efecto acaricida por lo que se ha utilizado para el control de Varroa destructor con una mortalidad de ácaros que oscila entre 70.63 % a un 56.25 % después de 3 semanas de tratamientos (Goswami & Khan, 2013), así mismo, se encontró un 98 % de mortalidad de Varroa destructor con aceite de canela a una concentración de 10 % (Kraus et al., 1994). Además, se ha comprobado que la canela funciona para estimular el aprendizaje de las abejas melíferas (Abramson et al., 2006). ...
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... Our results confirm the excellent antimicrobic properties of Cinnamomum oil, already successfully applied in vitro and in vivo against Paenibacillus larvae [35,[42][43][44], the ethiological agent of the american foulbrood, and against A. apis using both contact-dependent and contact-independent methods [30]. E-cinnamaldehyde was the major component present in the Cinnamomum oil used in our bioassays (79.3%). ...
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The use of natural substances such as essentials oils against bee pathogens is of great interest as an alternative to traditional methods based on synthetic compounds like antibiotics and fungicides, in order to minimize the risk of having toxic residues in hive products and to prevent the development of resistance phenomena. This study evaluated the inhibitory, fungicidal and sporicidal activity of ten essential oils extracted from aromatic plants against Ascosphaera apis, the etiological agent of chalkbrood, an invasive honey bee mycosis. The most effective essential oils were Thymus herba-barona, Thymus capitatus and Cinnamomum zeylanicum, which showed values of minimum fungicidal concentration and minimum sporicidal concentration ranging from 200 to 400 ppm. Carvacrol was the main component of Thymus capitatus and Thymus herba-barona oils, whereas cinnamic aldehyde prevailed in Cinnamomum zeylanicum oil. Further in-apiary studies will allow the evaluation of side effects on bees and residues in hive products.
... In order to improve the biological control of Apis mellifera L. diseases, cinnamon oil Cinnamomum zeylanicum was used against Paenibacillus larvae, then was evaluated in the laboratory and in a field (Gende et al., 2009). In addition, Antúnez et al., 2008 evaluated the effect of a Propolis ethanolic extract against P. larvae in lieu of chemical antibiotic and its positive effect. ...
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American foulbrood (AFB) Paenibacillus larvae and European foulbrood (EFB) Melissococcus plutonius are harmful for honey bee colonies. Many antibiotics were used, but mischief on Human healthy. Here, the natural products [Honey bees, Cinnamon. (Cinnamomum zeylanicum), Cloves (Syzygium aromaticum), Propolis, and Thymol (Thymus vulgaris)] were evaluated against AFB and EFB bacteria in laboratory and field. In laboratory Thymol had the highest effect on AFB with different concentrations and had a total mean of 3.37 ± 1.03 cm of circle around disc diffusion. In contrast, Thymol had the lowest effect on EFB with a total mean 0.33 ± 0.15 cm. The Cloves had the highest effect on EFB with a total mean of 4.50 ± 2.00 cm and fourth level effect on AFB with a total mean of 0.73 ± 0.20 cm. Propolis had the second level effect on AFB and EFB with total mean 1.15 ± 0.93 cm on AFB and 1.49 ± 0.95 cm on EFB. In Apiary, Thymol has the biggest change amount of brood development of 5.457 in each check time for AFB with a rate of 25.8% increase of capped brood through all the treated period contrast with EFB. On the other hand, Cloves had the biggest change amount of 5.371 of each check time treatment with a rate of 25.3% increase of capped brood through all treated periods. In addition, the mean weight of healthy pupae was 1.747 ± 0.172 mg. Pupae was infected by AFB weighed 1.101 ± 0.042 mg and pupae was infected with EFB recorded 1.344 ± 0.119 mg.
... Beekeepers, bee diet manufacturers, and researchers often add natural oils and other substances to pollen substitutes in an attempt to improve the health of honey bee colonies (Gende et al. 2009;Emsen and Dodologlu 2015). However, it is difficult to objectively measure the efficiency of such supplements in improving bee health. ...
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Among their natural defenses against pathogens and parasites, honey bees coat nest cavity surfaces with propolis. Consequently, they are able to economize on immune system activation, lowering energetic costs and improving longevity. However, the mechanisms through which propolis acts to protect bees are unknown. Here we show that 0.1% propolis fed in a pollen substitute diet greatly increases activation of antimicrobial peptide genes (defensin-1, abaecin, hymenoptaecin, and apidaecin) in bees injected with Escherichia coli, compared to infected bees fed the same diets without propolis. This increase was not seen in uninfected bees fed propolis. In addition to its protective role in the hive, propolis stimulates high-level expression of the immune system response in bees challenged with microorganisms. Whether this increase translates into improved disease control will require laboratory and field tests with pathogens.
... Későbbi vizsgálataik során a fahéjolaj MIC-értékét a kísérlet során használt oxitetraciklin antibiotikumhoz hasonló, 50 µg/ml koncentrációban határozták meg az általuk vizsgált P. larvae törzsekkel szemben. A fahéjolaj a betegség kezelésében az oxitetraciklinhez hasonló mértékben hatásosnak bizonyult mesterséges ráfertőzéses kísérlet során is (16). ...
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Background: American foulbrood (AFB) is the most important bacterial disease of honeybees with worldwide distribution, which causes high economic losses. The causative agent is P. larvae a Gram-positive, rod-shaped, motile, spore forming bacterium species. Usage of antibiotics for treatment of this disease is not allowed in most countries because of the residues in honey and other bee-products. Therefore, there is a great interest to find alternative and effective AFB-controlling natural substances, such as plant essential oils, which have natural antibacterial effect. Objectives: Antibacterial activity (Minimal Inhibitory Concentration) of a veterinary medical product called "NAF" (Chemor Ltd., Hungary) against 30 P. larvae strains isolated from different area of Hungary was determined. Strains originating from Hungarian apiaries were isolated and identified in 2015. Materials and Methods: Vegetative cells of P. larvae were grown on Columbia Agar supplemented with 10% defibrinated sheep blood. Bacterial suspension was set to MacFarland standard 0.5 that corresponds with 10⁵ CFU/ml bacterial density. Determination of MIC-valueswere carried out using broth dilution method in 48-well tissue culture plates. After the inoculation of the bacteria the tissue culture plates were incubated at 37°C for 48 hours. After the incubation period the bacterial growth was evaluated with naked eye. Results and Discussion: MIC range of NAF veterinary medical product was between 0.015 and 1.953 μl/ml in case of the investigated P. larvae strains. MIC50 and MIC90 values were both determined as 0.997 μl/ml. In vivo effect of the product should be carefully evaluated due to the poor information about the concentration of the active ingredients in the royal jelly. There-fore, conclusion about in vivo efficacy of the product should not be deducted, based on the in vitro susceptibility of the vegetative from of the pathogen.
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Trypanosomatid gut parasites are common in pollinators and costly for social bees. The recently described honey bee trypanosomatid Lotmaria passim is widespread, abundant, and correlated with colony losses in some studies. The potential for amelioration of infection by antimicrobial plant compounds has been thoroughly studied for closely related trypanosomatids of humans and is an area of active research in bumble bees, but remains relatively unexplored in honey bees. We recently identified several floral volatiles that inhibited growth of L. passim in vitro. Here, we tested the dose-dependent effects of four such compounds on infection, mortality, and food consumption in parasite-inoculated honey bees. We found that diets containing the monoterpenoid carvacrol and the phenylpropanoids cinnamaldehyde and eugenol at >10-fold the inhibitory concentrations for cell cultures reduced infection, with parasite numbers decreased by >90% for carvacrol and cinnamaldehyde and >99% for eugenol; effects of the carvacrol isomer thymol were non-significant. However, both carvacrol and eugenol also reduced bee survival, whereas parasite inoculation did not, indicating costs of phytochemical exposure that could exceed those of infection itself. To our knowledge, this is the first controlled screening of phytochemicals for effects on honey bee trypanosomatid infection, identifying potential treatments for managed bees afflicted with a newly characterized, cosmopolitan intestinal parasite.
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هذا الكتاب • يندرج نحل العسل تحت جنس Apis الذي يضم تسعة أنواع. وقد عُرف من سلالات نحل العسل العالمي أكثر من 24 سلالة. وتتكون طائفة نحل العسل من الملكة، والذكور، والشغالات. وتوجد مجموعة من الشروط يجب مراعاتها عند إنشاء المناحل ليكون مشروع المنحل ناجحًا. ويقوم النحال بمجموعة من العمليات النحلية التي تحتاجها الطائفة، مثل التشتية، وتغذية الطوائف، وتقسيم الطوائف أو ضمها، والتي يجب إجراؤها في مواعيدها بلا تأخير للمحافظة على قوة الطوائف والحصول على إنتاجية عالية. وتتعدد المنتجات التي من أجلها يُربى نحل العسل، وتشمل: العسل، وحبوب اللقاح وصمغ النحل (البروبوليس)، وشمع النحل، وسُم النحل، والغذاء الملكي، والملكات، وطرود النحل، هذا بالإضافة إلى دوره في تلقيح الأزهار وزيادة الإنتاج. ويُصاب نحل العسل أو أقراصه الشمعية بمجموعة من الآفات أو الأمراض التي تسبب للنحل أو أحد أطواره أو أقراصه الشمعية أضراراً بالغة. ليس هذا فحسب, بل أحياناً في حالة شدة الإصابة قد تؤدي إلى هلاك الطائفة أو الهجرة وترك الخلية والمنحل. ولكل آفة أو مرض أعراض وكذلك طرق الوقاية والعلاج منها. وهناك كثير من معوقات تربية نحل العسل في المملكة العربية السعودية، وعرض الكتاب لمجموعة من الوسائل المقترحة للنهوض بتربية نحل العسل في المملكة.
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The action of 21 essential oils against six strains of Bacillus larvae White, the causal agent of American foulbrood of honeybees, was studied in vitro. In a preliminary trial (table II) the effectiveness of either pure or diluted (1:5) oils was assessed by the diffusion agar method (Carta et al, 1989). The more effective ones were then tested in nutrient broth, at doses ranging from 50-400 mg/kg, to determine the minimal bactericide and sporicide concentrations. The most significant results were obtained with the oils of Citrus sinensis, Cinnamomun sp, Cuminum cyminum, Eugenia spp, Thymus vulgaris and reconstituted oil of Verbena (tables III and IV, fig 1). The same oils proved to be non-toxic for adult honeybees at the dose of 400 mg/kg. Cinnamomun oil was the most effective, with minimal bactericide and sporicide concentrations of 50 and 100 mg/kg respectively. This oil also gave positive results in controlling the American foulbrood in field trials, particularly in autumn and winter, when administered in semi-solid food (honey + caster sugar, 1:1.5) at a concentration of 400 mg/kg. In particular, the action of Cinnamomun oil on disease growth was evaluated in two distinct trials using four and five nuclei, respectively, obtained from the same colony (Apis mellifera ligustica Spin) so that each of them contained an approximately equal amount of adult bees, brood, honey and pollen beside a young queen (Liendenfelser, 1968). In both experimental trials, the worker brood was also carefully examined periodically by laboratory tests (Lloyd, 1986). The results showed that during autumn and winter, when the bees' consumption of the extra food is favoured by the environmental conditions (weather, scarcity or absence of flowers), Cinnamomun oil acts in vivo on B larvae. However, its use in the control of American foulbrood poses several practical problems including the time and the method of administration to take full advantage of the bactericidal properties found in vitro. If essential oils can be used against American foulbrood, the toxicological risks and the establishment of undesirable resistance factors associated with use of chemicals would be avoided.
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In honey bees (Apis mellifera L.), American foulbrood (AFB) is caused by the infection of the larvae and pupae with the bacterium Paenibacillus larvae subsp. larvae. The antibacterial effects of the essential oils of wild camomile (Tagetes minuta L.) and Andean thyme (Acantholippia seriphioides A. Gray) against different strains of P. larvae subsp. larvae were evaluated in vitro. The possible inhibitory effects of two emulsifiers of these oils (propylene glycol and soybean lecithin) were also assessed. Significant differences in antibacterial action were seen between the oils and emulsifiers (P < 0.01). Andean thyme had the strongest antibacterial effect; the minimum inhibitory and minimum bactericide concentrations were 200-250 mg l-1 and 300 mg l-1 respectively. An additional inhibitory effect was seen when propylene glycol was used as an emulsifier; no such extra effect was seen when soybean lecithin was employed. Additional key words: Acantholippia seriphioides, bacteria, honey bee, propylene glycol, soybean lecithin, Tage- tes minuta. Resumen
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American Foulbrood (AFB), caused by the spore-forming bacterium Paenibacillus larvae larvae, is the most serious disease of honeybees. Laboratory and field trials were conducted to evaluate the effectiveness of essential oils from savory, thyme, lemon-grass, and oregano, and blends of lemon-grass + thyme; lemon-grass + thyme + oregano + basil, and lemon-grass + thyme + basil in preventing and controlling infections of AFB in Apis mellifera colonies. The principal components of the essential oils used were determined by gas chromatography. LD 50 values for acute oral toxicity of the oils on adult bees verified that the essences were non-toxic or slightly toxic. Results from field trials indicate that neither the essences nor the blends were effective in the elimination of AFB clinical symptoms at any dose formulation or method of administration tested, whereas tylosin was highly effective in eliminating AFB clinical symptoms.
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The contact and oral acute toxicity of technical dimethoate to worker honey bees (Apis mellifera) was determined between May and October for 12 consecutive years, 1981–1992 (63 contact tests and 62 oral tests), using standard laboratory methods. The 24-h LD50 values ranged from 0.11 to 0.26 (mean 0.16) μg a.i./bee for contact toxicity, and from 0.11 to 0.33 (mean 0.18) μg a.i./bee for oral toxicity. The 48-h LD50 values were similar to the 24-h ones, indicating that there were no delayed toxic effects. There were no significant seasonal trends in contact or oral toxicity nor were there any consistent trends over the 12-year period. A comparison of British bees with a strain of New Zealand bees indicated that variability between individual colonies was greater than between the two strains. Published results from studies carried out elsewhere in the UK and in Germany are similar (LD50 values within a factor of about two of the mean values presented here). It is concluded that technical dimethoate is suitable as a reference compound for laboratory toxicity tests with honey bees.
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American foulbrood (AFB) is a severe bacterial brood disease of honey bees. AFB is lethal to bee colonies if treatment is not carried out. The disease is distributed worldwide and can cause substantial economic losses. Foulbrood was first described in 1769 and although AFB has been in focus for decades it has recently become an increasing problem.
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The essential oil obtained by hydrodistillation from the fruits of Pimpinella anisum L. (green anise) and Foeniculum vulgare Miller (fennel) were analyzed by GC and GC/MS and physicochemical properties. The oils of P. anisum and F. vulgare were found to be especially rich in (E)-anethole, 96.3% and 92.7%, respectively. The MICs were determined by the tube dilution method against Paenibacillus larvae. The oils showed MICs values were 300 µg/mL and 250 μg/mL for P. anisum and F. vulgare, respectively. Both oils presented great similarity in physiochemical properties values and antimicrobial activity.