główne kierunki zainteresowania badawczego Pracowni Chorób Owadów Użytkowych SGGW

Ginięcie rodzin pszczelich, wirusy i nosema jako główne kierunki zainteresowania badawczego Pracowni Chorób Owadów Użytkowych SGGW Grażyna Topolska Wydział Medycyny Weterynaryjnej SGGW

Grochów do 2001r

Ursynów

1992 wykrycie 7 wirusów pszczół w Polsce

Test 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 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 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 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 importance increased when the mite Varroa destructor t arrived in Europe and was found to be a vector of many viruses and an activator of virus infections. At the beginning of the 21 st 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 E., Kołodziejekł dijkj., Rusvai M., Topolska lk 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 Ladder, 1 BQCV, 2 SBV, 3 ABPV, 4 DWV, 5 CBPV

Topolska G. (2008) Zakażenia wirusowe czerwiu matecznego oraz matek pszczelich w dziesięciu pasiekach hodowlanych w Polsce [Virus infections of queen brood and queen bees in ten queen rearing apiaries in Poland]. 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 A B

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

Masowe ginięcie rodzin pszczelich w USA straty rodzin w % 40 35 30 32 36 29 34 30 25 20 15 10 5 0 2007 2008 2009 2010 2011 18

2006 r. USA CCD Colony Collapse Disorder Zespół ginięcia rodzin pszczelich Objawy nagłe słabnięcie rodzin ginięcie ę pszczół poza ulem zostaje garstka pszczół z matką na plastrach z czerwiem i jedzeniem pozostały pokarm nie jest rabowany 19

Badanie rozmiaru strat rodzin pszczelich

Straty rodzin w Polsce 20 18 16 14 12 10 8 6 4 2 0 Straty rodzin w % 2006/2007 2007/2008 2008/2009 2009/2010 2009/2010 korygowane* 2010/2011 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. It was commonly Suwałki Lane 0 100 bp DNA ladder (1995) 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 Szczecin was proved that in the US it had been present in apiaries since at least 1996 (Chen et al. 2007) and Warsaw (1995,1996) in Finland since 1998 (Paxton et al. 2007). It has Poznań Biała Podlaska been diagnosed by the team of Professor Piotrków Trybunalski (1996) Aranazu Meana and Dr Mariano Hies (1996) 300 bp Lubin (performing PCR analysis), that Nosema ceranae Ostrowiec (1995) 200 bp Świętokrzyski was present in each of ten dead bee samples (from four different Polish apiaries) sent to them Dzięgielów in 2007. In WULS samples collected between a) Lanes: 1, 2, 3, 4, 5, 6 and 9 samples from 1995; Lanes: 7, 8 (1996) 1994-1995 in different parts of Poland were and 10 samples from 1996; lane 11 positive control examined. Nosema spores were found in 92% of (Nosema apis and Nosema ceranae); lane 12 negative control the colonies examined (which appeared to be (PCR grade water). healthy). strong signal weak signal n this work historical samples and present 0 1 2 3 4 5 6 7 Map 1) Cities from which the samples were samples from Poland were investigated for the collected and examined by PCR. presence of Nosema ceranae. Stars indicate the results obtained during electrophoresis of PCR amplicons. Materials and methods Numbers in brackets indicate the year when We examined 25 dead bee samples collected from 300 bp samples were collected. apparently healthy colonies in the years 1994-1996 1996 (3 - in 1994, 16 - in 1995, 6 - in 1996) and 432 200 bp samples, collected between 1 st of December 2007 and 15 th of March 2009, from colonies in which b) Lanes: 1, 2 and 3 samples from 1995; lanes 4 and 5 increased mortality of bees or disappearing of bees samples from 1996; lane 6 positive control (Nosema apis was observed (apiaries from different parts of and Nosema ceranae); lane 7 - negative control (PCR grade Poland). water). The samples were investigated microscopically (Topolska G., Kaprzak S. 2007; Topolska et al 0 1 2 3 4 5 6 7 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 1 st of December 2007 and 15 th of March 2009, Nosema 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.) 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). 300 bp 200bp 0 1 2 3 4 5 6 7 Fig. 2) Result of the electrophoresis of the amplicons produced by PCR of the samples collected between 1 st of December 2007 and 15 th 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). Grant nr N308010 32/1204 from the Ministry of Science and Higher Education in Warsaw Conclusions 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 that Nosema ceranae is present in most colonies suffering from nosemosis in Poland. 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 idi 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. 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.

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 andinnortherncountries,stilln. 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. 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. Colonies No. of colonies Live bees Dead winter 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 2009 2010 2011 1 1sw 1sw 1sp 3 2sw 1sw 1sp 5 1sw 1sw 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. % of colonies Graph 1. Nosema species determination during the two seasons in colonies type II 80% 60% 40% 20% 0% 2009 2010 mixed infection Nosema apis Nosema ceranae <0.01 mln spores=pcr undetectable of infected bees % 100% 80% 60% 40% 20% 0% Graph 2. Percentage of infected bees in colonies type I in the following 3 springs 2009 2010 2011 hive 1 hive 3 hive 5 num mber of spores in millions/bee 40 30 20 10 0 Spring 2009 Graph 3. The level of infection in type I colonies Graph 4. Percentage of type II colonies with different level of Graph 5. Percentage of type II colonies with different levels of infection (season) from 15.1 mln infection during the two winters 60% spores from 15.1 mln 40% 50% 5.1 15 mln spores 40% spores 30% 5.1 15 mln spores 30% 0.01 5 mln 20% 0 5 mln spores 20% spores 10% less than 0.01 10% 0 spores 0% mln spores 0% 2009 2010 2011 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. Spring 2010 Spring 2011 Autumn 2009 Autumn 2010 Hive 1 Hive 5 Hive 3

Przebieg zakażenia Nosema ceranae w Polsce 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)

Przebieg zakażenia Nosema ceranae w Polsce z uwzględnieniem wpływu towarzyszących ą y zakażeń wirusowych Wirus choroby czarnych mateczników (black queen cell virus) Dicistroviridae Wirus Y pszczół Wirus włókienkowy (filamentous virus) Baculoviridae? Analiza próbek terenowych (PCR, RT PCR, AGID, mikroskopia k i elektronowa) lkt Badania laboratoryjne: zakażanie pszczół Nosema spp. + wirusami (analizy real time PCR, real time RT PCR)