Ginięcie rodzin pszczelich wirusy i nosema jako Ginięcie rodzin

Ginięcie rodzin pszczelich, wirusy i nosema
Ginięcie
rodzin pszczelich wirusy i nosema jako jako
główne kierunki zainteresowania badawczego Pracowni Chorób Owadów Użytkowych SGGW
Grażyna Topolska Wydział Medycyny Weterynaryjnej
Wydział Medycyny Weterynaryjnej SGGW
Grochów do 2001r
Grochów do 2001r
Ursynów
1992‐wykrycie 7 wirusów pszczół w Polsce
Test AGID
Test AGID
Produkcja surowic do AGID
Produkcja surowic do AGID
Annals of Warszaw Agricultural University – SGGW
Animal Science No 63, 2009.
(Ann. Warsaw Agricult. Univ. – SGGW, Anim. Sci. No 63, 2009)
The investigation of bee virus infections in Poland
GRAŻYNA TOPOLSKA, KATARZYNA KRZYŻAŃSKA*, ALEKSANDRA HARTWIG,
ANNA GAJDA
University of Life Sciences – SGGW Faculty of Vetrinary Medicine, Department of Clinical Sciences, Laboratory of Bee
Diseases
*Actual: Urząd Rejestracji Produktów Leczniczych, Wyrobów Medycznych i Produktów Biobójczych,
Abstract: In 1995-1996 adult dead bee samples were collected from April to September from apparently healthy colonies in
nine apiaries located in different parts of Poland. The samples were tested by agar gel immunodiffusion (AGID) for black
queen cell
ll virus
i (BQCV)
(BQCV), acute
t bee
b paralysis
l i virus
i (ABPV),
(ABPV) chronic
h i bee
b paralysis
l i virus
i (CBPV) andd sacbrood
b d virus
i (SBV),
(SBV)
also by microscopy for Nosema spores and by electron microscopy for filamentous virus (FV). Nosema was a very common
infection in the investigated apiaries. It was detected in 80% of tested colonies in 1995 and in 79% of colonies in 1996. FV
and BQCV were found in all the apiaries, respectively in 70 % and 60 % of investigated colonies. The percentage of samples
infected with FV and BQCV was very high in the period April – June, with the peak in May. ABPV was found in six apiaries
– located in the area where a marine climate predominates over a humid continental one - in 24% of investigated colonies,
mainly in samples collected from July to September. SBV was detected in five apiaries (approximately in 10% of colonies),
mainly in April and May. CBPV also occurred in five apiaries (15% of colonies), mainly in May – June. In three apiaries five
viruses and in the other three – four viruses per apiary were found. In about 6% of colonies infection by at least three viruses
was detected.
Key words: honey bee, viruses, Poland, 1995-1996
INTRODUCTION
According to the latest discoveries of American scientists viruses can be one of the main
causes of CCD – Colony Collapse Disorder which since 2006 has caused each year the
disappearance of a considerable number of bee colonies in the USA. Johnson and co-authors
(2009) report that “The
The reduced protein synthetic capabilities that would accompany
ribosomal hijacking by multiple picorna-like viruses would leave bees unable to respond to
additional stresses from pesticides, nutrition, or pathogens”. Fifty years ago only two virus
infections attracted the attention of scientists - chronic bee paralysis virus and sacbrood virus
(Bailey et,al., 1963). 20 years later about 16 honey bee viruses were known and their
i
importance
t
increased
i
d when
h the
th mite
it Varroa
V
destructor
d t t arrived
i d in
i Europe
E
andd was found
f d to
t be
b
a vector of many viruses and an activator of virus infections. At the beginning of the 21st
century molecular biology methods (RT-PCR) became the main tool in investigating the most
important of these viruses (Benjeddou et al. 2001; Grabensteiner et al. 2001) . Since then new
t h
b
i
i di ll b t d t ti
f
ti l
i
i
i
Współpraca zagraniczna i wprowadzenie Rt‐PCR
•
Bakonyi T., Grabensteiner
k i G b
i
E., Kołodziejek
ł d i j k J., Rusvaii M., Topolska G., Ritter W., lk G i
Nowotny N. (2002) Phylogenic Analysis of Acute Bee Paralysis Virus Strains. Appl. Environ. Microbiol. 68: 6446‐6450
•
Berényi O., Bakonyi T., Derakhshifar I, Köglberger H., Topolska G., Ritter W., Pechhacker H., Nowotny N. (2007) Phylogenetic Analysis of Deformed Wing Virus Genotypes from Diverse Geographic Origins Indicates Recent Global Distribution of the Virus. Appl. Environ. Microbiol., 73: 3605‐3611
•
Tapaszti Z., Forgách, Kővágó C., Topolska G., Nowotny N., Rusvai M., Bakonyi T. (2009) Genetic analysis and phylogenetic comparison of Black queen cell virus genotypes. Veterinary Microbiology
•
•
Wyniki elektroforezy produktów PCR : 0 Wyniki
elektroforezy produktów PCR : 0 ‐ GeneRuler 1000 bp DNA Ladder, 1000 bp DNA Ladder
1 ‐ BQCV, 2 ‐ SBV, 3 ‐ ABPV, 4 ‐ DWV, 5 ‐ CBPV
• Topolska G. (2008) Zakażenia wirusowe g
p
czerwiu matecznego oraz matek pszczelich w dziesięciu pasiekach hodowlanych w Polsce [Virus infections of queen
[Virus infections
of queen brood and queen
and queen
bees in ten queen rearing apiaries in Poland]. Wydawnictwo SGGW ]
Wydawnictwo SGGW.]
Zakażenia wirusowe czerwiu matecznego oraz matek pszczelich
Wirus choroby czarnych mateczników najczęstsza przyczyną zamierania larw matecznych w pasiekach hodowlanych w Polsce
Wyniki elektroforezy produktów PCR : 0 ‐
y
yp
GeneRuler 100 bp DNA Ladder, p
,
1 – 10 larwy z zamarłych mateczników
Zakażenia wirusowe czerwiu matecznego oraz matek pszczelich
W „zamarłych matecznikach” zakażonych BQCV najczęściej stwierdzano obecność lekko pożółkłych poczwarek
A
B
Spory Nosema apis (A) i Nosema ceranae (B)
Masowe ginięcie rodzin pszczelich w USA
straty rodzin w % 40
35
36
34
32
30
29
30
25
20
15
10
5
0
2007
2008
2009
2010
2011
18
2006 r.
2006 r.‐‐ USA
CCD –C
CCD –
CCD Colony Collapse Disorder
l
C ll
Di d
Zespół ginięcia rodzin pszczelich
Objawy
• nagłe słabnięcie rodzin
• gginięcie pszczół poza ulem
ę p
p
• zostaje garstka pszczół z matką na plastrach z czerwiem i jedzeniem
• pozostały pokarm nie jest rabowany
19
Badanie rozmiaru strat rodzin pszczelich
Badanie rozmiaru strat rodzin pszczelich
Straty rodzin w Polsce
Straty rodzin w Polsce
Straty rodzin w %
20
18
16
14
12
10
8
6
4
2
0
2006/2007
2007/2008
2008/2009
2009/2010
2009/2010 2010/2011
korygowane*
22
Coloss questionnaire results
Nosema ceranae has been present in Poland
since at least 1995
Grażyna Topolska, Anna Gajda
Warsaw University of Life Sciences, Faculty of Veterinary Medicine, Department of Clinical Sciences, Laboratory of Bee
Diseases.
Introduction
Fig. 1) Results of the electrophoresis of the amplicons produced
Nosema ceranae infection is widely present in
by PCR of samples from 1994 -1996.
apiaries all over the world.
world It was commonly
L
Lane
0 – 100 bbp DNA lladder
dd
believed that this parasite had infected Apis
0 1 2 3 4 5 6 7 8 9 10 11 12
mellifera only in recent years. However, lately it
was proved that in the US it had been present in
apiaries since at least 1996 (Chen et al. 2007) and
in Finland since 1998 (Paxton et al. 2007). It has
been diagnosed by the team of Professor
Aranazu Meana and Dr Mariano Hies
300 bp
(performing PCR analysis), that Nosema ceranae
200 bp
was present in each of ten dead bee samples
(from four different Polish apiaries) sent to them
p collected between
in 2007. In WULS samples
a)) Lanes: 1,, 2,, 3,, 4,, 5,, 6 and 9 – samples
p from 1995;; Lanes: 7,, 8
and 10 – samples from 1996; lane 11 – positive control
1994-1995 in different parts of Poland were
examined. Nosema spores were found in 92% of (Nosema apis and Nosema ceranae); lane 12 – negative control
(PCR grade water).
the colonies examined (which appeared to be
healthy).
0 1 2 3 4 5 6 7
•n this work historical samples and present
samples from Poland were investigated for the
presence of Nosema ceranae.
Materials and methods
We examined 25 dead bee samples collected from
apparently
tl healthy
h lth colonies
l i in
i the
th years 1994-1996
1994 1996
(3 - in 1994, 16 - in 1995, 6 - in 1996) and 432
samples, collected between 1st of December 2007 and 15th of March 2009, from colonies in which
increased mortality of bees or disappearing of bees
was observed (apiaries from different parts of
Poland).
The samples were investigated microscopically
(Topolska G., Kaprzak S. 2007; Topolska et al
2008) and by PCR (OIE Manual For Terrestrial
Animals; Higes et al. 2006). The preliminary
treatment of the samples with sodium hypochlorite
( Fedorko et al. 1995) was introduced due to the
fact that dead bees (containing much more bacteria
and melanine than live bees) were examined. The
bleach was supposed to eliminate the bacteria and
clear up the sample. The amplicon sizes for
Nosema apis and Nosema ceranae were 321 and
218-219 bp respectively.
Results
The sequences characteristic for N. ceranae were
found in two samples from 1995 - from a Warsaw
apiary ( Fig. 1a) and one sample from 1996 - from
Biała Podlaska (Fig. 1b).
During the electrophoresis of PCR products a
weak signal was obtained in the case of 4 other
samples from 1995 and 2 samples from 1996
(Fig. 1a). A very weak signal was obtained also in
the case of two samples from 1994, however a
more sensitive method should be used to confirm
the presence of N. ceranae in these samples.
In 306 (68%) samples, collected between 1st of December 2007 and 15th of March 2009, Nosema
spores were detected
detected. In 250 (82%) of these
samples N. ceranae was present.
The comparison of results of investigations done
with and without the use of sodium hypochloride
indicated that this preliminary treatment was
useless – the signal in electrophoresis was
weaker.( Fig. 2.)
300 bp
Suwałki
(1995)
Szczecin
Poznań
Lubin
(1995)
Warsaw
(1995,1996)
Biała Podlaska (1996)
Piotrków Trybunalski
(1996)
Ostrowiec Świętokrzyski
Dzięgielów
(1996)
‐ strong signal
‐ weak signal
Map 1) Cities from which the samples were
collected and examined by PCR.
Stars indicate the results obtained during
electrophoresis of PCR amplicons.
Numbers in brackets indicate the year when
samples were collected.
200 bp
Conclusions
b) Lanes: 1, 2 and 3 – samples from 1995; lanes 4 and 5 –
samples from 1996; lane 6 – positive control (Nosema apis
and Nosema ceranae); lane 7 - negative control (PCR grade
water).
0
1
2
3
4
5
6
7
1. Nosema ceranae was already present in Polish
apiaries in 1995 and 1996.
2. We were not able to show the presence of
Nosema ceranae in Poland in 1994, however
the very weak signal obtained during
electrophoresis of PCR products suggests that
that Nosema ceranae might have been present
in Poland before 1995.
3 The examination of recent samples showed
3.
that Nosema ceranae is present in most
colonies suffering from nosemosis in Poland.
300 bp
200 bp
c) Lanes: 1, 2 and 3 – samples from 1994: lane 4 – sample
from 1995; lane 5 – sample from 1996; lane 6 – positive
control (Nosema apis and Nosema ceranae); lane 7 –
negative control (PCR grade water).
0
0 1 2 3 4 5 6 7
1
2
3
4
5
6
7
300 bp
200bp
Fig. 2) Result of the electrophoresis of the amplicons
produced by PCR of the samples collected between 1st of th of March 2009.
December 2007 and 15th
December 2007 and 15
of March 2009
Lane 0 – 100 bp molecular marker: lanes 1, 2, 3 and 4 –
samples treated with sodium hypochloride; lane 5 – the
previous sample without the treatment of sodium
hypochloride; lane 6 – negative control (PCR grade water);
lane 7– positive control (Nosema apis and Nosema ceranae).
References
1. Chen Y., Evans J., Smith I., Pettis J.: Nosema
ceranae is a long-present and wide spread
microsporidian infection of the European honey bee
(Apis mellifera) in the United States. Journal of
Invertebrate Pathology 97 (2008), 186-188.
2. Higes M., Martin R., Meana A.: Nosema ceranae, a
new microsporidian
i
idi parasite
it in
i honeybees
h
b
in
i Europe.
E
Journal of Invertebrate Pathology 92 (2006), 93-95.
3. Paxton R., Klee J., Korpela S., Fries I.: Nosema
ceranae has infected Apis mellifera in Europe since
at least 1998 and may be more virulent than Nosema
apis. Apidologie 38 (2007), 558- 565.
4. Topolska G., Gajda A., Hartwig A.: Polish honey
bee colony-loss during the Winter of 2007/2008.
Journal of Apicultural Science Vol. 52, No. 2, 2008,
95-104.
5. Topolska G., Kasprzak S.: First cases of Nosema
ceranae infection in bees in Poland. Medycyna
Weterynaryjna 2007, 63 (11) Supplement.
6. Fedorko D., Nelson N., Cartwright C.: Identification
of Microsporidia in stool specimens by using PCR
and restriction endonucleases. Journal of Clinical
Microbiology, 1995, vol. 33, No. 7, p. 1739- 1741.
Grant nr N308010 32/1204 from the Ministry of Science and Higher Education in Warsaw
The course of Nosema ceranae infection in Poland
Anna GAJDA, Grażyna TOPOLSKA, Warsaw University of Life Sciences, Faculty of Veterinary Medicine, Department
of Pathology and Veterinary Diagnostics, Laboratory of Bee Diseases.
Introduction
In Spain Nosema cearanae is considered as the main cause of great colony losses. However the course of N. ceranae infection is suspected to differ greatly between
different geographical locations, hence different climatic conditions. Scientific reports suggest, that in southern European countries predominant species is N. ceranae
and in northern countries, still N. apis. The aim of our investigation is to observe and determine the course of N. ceranae infection in Polish climatic conditions.
Material and methods
Colonies of two types were investigated:
I ‐ colonies from WULS with pure N. ceranae infection from at least 2007, II‐ colonies formed for COLOSS – GEI experiment with predominant, usually well developed,
mixed N. apis and N. ceranae infection
Kind of bee samples: Type I colonies – foragers at the hive entrance and dead bees from winter debris. Type II colonies– in the spring of 2009 foragers at the hive
entrance later ‐ bees from the outer comb .
entrance,
Methods of Nosema spp. detection and identification: described in OIE Manual standardised method (the number of bees changed to 30) and PCR also recommended
by OIE.
Dead winter bees
Colonies No. of colonies Live bees
Type I 3 No. of spores/bee % of infected bees Nosema species
Type II 48
No. of spores/bee Nosema species No. of spores/bee Table 1. Data gathered from the colonies
Colony
1
3
5
2009
1sw
2sw
1sw
2010
1sw
1sw
1sw 2011
1sp
1sp
2sw
Table 2. Number of swarms (sw) and splits
(sp) in colonies of type I
Results
Type I colonies: The number of spores per forager bee was, in all the colonies, in all the years of investigation smaller in autumn than in spring. It was also smaller in the
second year of observation than in the first year (graph 3.). Although percentage of infected bees was the highest in 2009 and 2011 (graph 2.). Several swarms and splits
were formed (Tab. 2.).
Type II colonies– as the experiment went on the percentage of less infected bees grew bigger (graph 4. and 5.). Also it becme evident, that are succesively dissapearing,
replaced by pure N. ceranae infections (graph 1.). in 33% of the colonies in which in 2009 N. apis or N. apis + N. ceranae were detected, in 2010 only N. ceranae was
found. Also In 2009, the mixed infections were more common (63% of colonies), whereas, in 2010, mixed infections were found in only 20% of examined colonies.
Investigation of dead bees collected from the hive bottom boards at the end of two winters (2009/2010 and 2010/2011) suggests, that in 64% of the colonies the level of
infection increased, while in 26% it decreased and in 9% it did not change.
Graph 2. Percentage of infected bees in colonies type I in the following 3 springs
Graph 3. The level of infection in type I colonies
num
mber of spores in millions/bee
Graph 1. Nosema species determination during the two seasons in colonies type II
40
100%
% of infected bees
% of colonies
80%
60%
40%
20%
0%
2009
2010
20
60%
40%
20%
2009
hive 1
2010
50%
hive 3
5.1‐15 mln spores
40%
30%
0.01‐5 mln spores
20%
10%
less than 0.01 mln spores
0%
2010
Hive 5
Hive 5
0
2011
Hive 3
Spring 2009
2011
Spring 2010
Spring 2011
hive 5
Graph 4. Percentage of type II colonies with different level of infection (season)
from 15.1 mln spores
2009
10
0%
mixed infection
Nosema apis
Nosema ceranae
<0.01 mln spores=PCR undetectable
60%
30
80%
Hive 1
Autumn 2009
Autumn 2010
Graph 5. Percentage of type II colonies with different levels of infection during the two winters
20%
from 15.1 mln spores
5.1‐15 mln spores
0‐5 mln spores
10%
0 spores
40%
30%
0%
2009/2010
2010/2011
Discussion and conclusions The results suggest, that although Nosema ceranae seems to be a dangerous pathogen in some regions of the world, in Poland it is not that big threat. The infection levels high in the beginning of the investigation now are fairly low. It may be due to proper beekeeping practices, such as intensive replacement of combs. Also it became clear, that the course of infection is very different in Poland than in Spain. The examined Polish colonies are still alive (5th year of infection) and with confirmed N. ceranae infection (Spanish bees die in the second year of infection). Moreover Polish colonies swarm, which is not seen in Spain. It also seems that N. ceranae is
succesively replacing N. apis in examined colonies.
Przebieg zakażenia Nosema ceranae
w Polsce
• Nosema
Nosema ceranae wypiera w pasiekach polskich Nosema ceranae wypiera w pasiekach polskich Nosema
apis
• Zakażenie w naszym klimacie nie oznacza śmierci rodzin w ciągu 2 lat
• Stopień zakażenia w poszczególnych latach nie wykazuje stałej tendencji wzrostowej (może zarówno wzrastać jak i maleć)
l ć)
Przebieg zakażenia Nosema ceranae
w Polsce z uwzględnieniem wpływu
w Polsce z uwzględnieniem wpływu towarzyszących zakażeń wirusowych
y ą y
y
•
Wirus choroby czarnych mateczników (black queen cell virus) Dicistroviridae
•
Wirus Y pszczół
Wirus Y pszczół
•
Wirus włókienkowy (filamentous virus) Baculoviridae?
• Analiza próbek terenowych (PCR, RT‐PCR, AGID, mikroskopia elektronowa)
ik k i l kt
)
• Badania laboratoryjne: zakażanie pszczół Nosema
spp. + wirusami (analizy ‐ real time PCR, real time RT‐PCR)