Ginięcie rodzin pszczelich wirusy i nosema jakoGinięcie rodzin pszczelich, wirusy i nosema jako główne kierunki zainteresowania badawczego 

Pracowni Chorób Owadów Użytkowych SGGW

Grażyna Topolska 

WydziałMedycyny WeterynaryjnejWydział Medycyny Weterynaryjnej 

SGGW

Grochów do 2001rGrochów do 2001r

UrsynówUrsynów

1992‐wykrycie 7 wirusów pszczół w Polsce

Test AGIDTest AGID

Produkcja surowic do AGIDProdukcja 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 BeeDiseases *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

ll i (BQCV) t b l i i (ABPV) h i b l i i (CBPV) d b d i (SBV)queen cell virus (BQCV), acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and sacbrood virus (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 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 maincauses of CCD – Colony Collapse Disorder which since 2006 has caused each year thedisappearance of a considerable number of bee colonies in the USA. Johnson and co-authors(2009) report that “The reduced protein synthetic capabilities that would accompany(2009) report that The reduced protein synthetic capabilities that would accompanyribosomal hijacking by multiple picorna-like viruses would leave bees unable to respond toadditional stresses from pesticides, nutrition, or pathogens”. Fifty years ago only two virusinfections 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 theiri t i d h th it V d t t i d i E d f d t bimportance increased when the mite Varroa destructor arrived in Europe and was found to bea 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 mostimportant 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

k i G b i ł d i j k i l k G i• Bakonyi T., Grabensteiner E., Kołodziejek J., Rusvai M., Topolska G., Ritter W., 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 ‐ GeneRuler 1000 bp DNA LadderWyniki elektroforezy produktów PCR : 0  GeneRuler 1000 bp DNA Ladder, 

• 1 ‐ BQCV, 2 ‐ SBV, 3 ‐ ABPV, 4 ‐ DWV, 5 ‐ CBPV

• Topolska G. (2008) Zakażenia wirusowe czerwiu matecznego oraz matek pszczelich w g pdziesięciu pasiekach hodowlanych w Polsce [Virus infections of queen brood and queen[Virus infections of queen brood and queenbees 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 ‐ GeneRuler 100 bp DNA Ladder, y y p 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

AA

B

Spory Nosema apis (A) i Nosema ceranae (B)

Masowe ginięcie rodzin pszczelich w USA

363435

40

straty rodzin w %  

3229

34

30

25

30

35

20

25

10

15

0

5

2007 2008 2009 2010 20112007 2008 2009 2010 2011

18

2006 r.2006 r.‐‐ USAUSACCDCCD C l C ll Di dC l C ll Di dCCD CCD ––Colony Collapse DisorderColony Collapse DisorderZespół ginięcia rodzin pszczelichZespół ginięcia rodzin pszczelich

ObjawyObjawy•• nagłe słabnięcie rodzinnagłe słabnięcie rodzin•• ginięcie pszczół poza ulemginięcie pszczół poza ulemg ę p pg ę p p•• zostaje garstka pszczół z matką na plastrach z czerwiem i zostaje garstka pszczół z matką na plastrach z czerwiem i 

jedzeniemjedzeniem•• pozostały pokarm nie jest rabowanypozostały pokarm nie jest rabowany

19

Badanie rozmiaru strat rodzin pszczelichBadanie rozmiaru strat rodzin pszczelich

Straty rodzin w PolsceStraty rodzin w Polsce

1820

Straty rodzin w %

12141618

681012

0246

02006/2007 2007/2008 2008/2009 2009/2010 2009/2010 

korygowane*2010/2011

22

Coloss questionnaire resultsColoss questionnaire results

Nosema ceranae has been present in Poland Nosema ceranae has been present in Poland since at least 1995

Introduction

Nosema ceranae infection is widely present in apiaries all over the world It was commonly

Grażyna Topolska, Anna GajdaWarsaw University of Life Sciences, Faculty of Veterinary Medicine, Department of Clinical Sciences, Laboratory of Bee Diseases.

Fig. 1) Results of the electrophoresis of the amplicons produced by PCR of samples from 1994 -1996.L 0 100 b DNA l dd Suwałkiapiaries all over the world. It was commonly

believed that this parasite had infected Apis 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(performing PCR analysis), that Nosema ceranaewas present in each of ten dead bee samples (from four different Polish apiaries) sent to them in 2007. In WULS samples collected between a) Lanes: 1, 2, 3, 4, 5, 6 and 9 – samples from 1995; Lanes: 7, 8

0       1       2        3         4        5        6          7        8         9      10      11    12

300 bp

200 bp

Lane 0 – 100 bp DNA ladder Suwałki

(1995)

Warsaw

(1995,1996)Biała Podlaska  

(1996)Piotrków Trybunalski

(1996)

Dzięgielów

(1996)

Lubin

(1995)

Szczecin

Ostrowiec Świętokrzyski

Poznań

p1994-1995 in different parts of Poland were examined. Nosema spores were found in 92% of the colonies examined (which appeared to be healthy).•n this work historical samples and present samples from Poland were investigated for the presence of Nosema ceranae.

Materials and methodsWe examined 25 dead bee samples collected from

tl h lth l i i th 1994 1996

) , , , , , p ; ,and 10 – samples from 1996; lane 11 – positive control (Nosema apis and Nosema ceranae); lane 12 – negative control (PCR grade water).

0         1            2            3            4             5            6           7

300 bp

Map 1) Cities from which the samples were collected and examined by PCR.Stars indicate the results obtained duringelectrophoresis of PCR amplicons.Numbers in brackets indicate the year whensamples were collected.

(1996)

‐ strong signal ‐ weak signal

apparently healthy colonies in the years 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

Conclusions1. 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

b) Lanes: 1, 2 and 3 – samples from 1995; lanes 4 and 5 –samples from 1996; lane 6 – positive control (Nosema apisand Nosema ceranae); lane 7 - negative control (PCR grade water).

200 bp

0 1 2 3 4 5 6 7

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.

3. The examination of recent samples showed that Nosema ceranae is present in most colonies suffering from nosemosis in Poland.

ResultsThe sequences characteristic for N. ceranae were found in two samples from 1995 - from a Warsaw

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 i idi it i h b i E

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).

300 bp

200 bp

0 1 2 3 4 5 6 7apiary ( Fig. 1a) and one sample from 1996 - from Biała Podlaska (Fig. 1b).During the electrophoresis of PCR products aweak 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, Nosemaspores were detected In 250 (82%) of these

new microsporidian parasite in honeybees in Europe. 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.

0          1           2           3          4          5         6           7

200bp

300 bp

Fig. 2) Result of the electrophoresis of the amplicons produced by PCR of the samples collected between 1st of December 2007 and 15th  of March 2009

Grant nr N308010 32/1204 from the Ministry of Science and Higher Education in Warsaw

spores were 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.)

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.

December 2007 and 15th 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).

The course of Nosema ceranae infection in PolandAnna GAJDA, Grażyna TOPOLSKA, Warsaw University of Life Sciences, Faculty of Veterinary Medicine, Departmentof Pathology and Veterinary Diagnostics, Laboratory of Bee Diseases.

IntroductionIn 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 betweendifferent geographical locations, hence different climatic conditions. Scientific reports suggest, that in southern European countries predominant species is N. ceranaeand 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 methodsColonies 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 infectionKind 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 hiveentrance later bees from the outer comb

Colonies No. of colonies  Live bees Dead winter bees

Type I  3  No. of spores/bee  % of infected bees Nosema speciesType II  48 No. of spores/bee  Nosema species No. of spores/bee 

entrance, later ‐ bees from the outer comb .Methods of Nosema spp. detection and identification: described in OIE Manual standardised method (the number of bees changed to 30) and PCR also recommendedby OIE.

Colony 2009 2010 20111 1sw 1sw 1sp3 2sw 1sw 1sp5 1sw 1sw  2sw

Table 2. Number of swarms (sw) and splits(sp) in colonies of type I

Results

Table 1. Data gathered from the colonies

ResultsType 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 thesecond 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 splitswere 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 wasfound. 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 ofinfection increased, while in 26% it decreased and in 9% it did not change.

Hive 5

10

20

30

40

mbe

r of spo

res in m

illions/bee

Graph 3. The level of infection in type I colonies

20%

40%

60%

80%

% of colon

ies

Graph 1. Nosema species determination during the two seasons in colonies type II

20%

40%

60%

80%

100%

of infected

 bees

Graph 2. Percentage of infected bees in colonies type I in the following 3 springs

Hive 1

Hive 3

Hive 50

Spring 2009

Spring 2010

Spring 2011

Autumn 2009

Autumn 2010

num

60%

Graph 4. Percentage of type II colonies with different level of infection (season) from 15.1 mln 

spores40%

Graph 5. Percentage of type II colonies with different levels of infection during the two winters

from 15.1 mln 

0%2009 2010

mixed infectionNosema apisNosema ceranae<0.01 mln spores=PCR undetectable

0%

20%

2009 2010 2011

% 

hive 1 hive 3 hive 5

Discussion and conclusions 

0%

10%

20%

30%

40%

50%

2009 2010 2011

5.1‐15 mln spores

0.01‐5 mln spores

less than 0.01 mln spores

0%

10%

20%

30%

40%

2009/2010 2010/2011

spores5.1‐15 mln spores0‐5 mln spores

0 spores

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 ceranaew Polsce

• Nosema ceranae wypiera w pasiekach polskich Nosema• 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 

l ć)maleć)

Przebieg zakażenia Nosema ceranaew Polsce z uwzględnieniem wpływuw Polsce z uwzględnieniem wpływu towarzyszących zakażeń wirusowychy 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, ik k i l kt )mikroskopia elektronowa)

• Badania laboratoryjne: zakażanie pszczół Nosemaspp. + wirusami (analizy ‐ real time PCR, real time RT‐PCR)