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EurBee board Dorothea Brückner, Germany Norberto Milani, Italy Robert Paxton, Great Britain Dalibor Titěra, Czech Republic Bernard Vaissiere, France Program consultant


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Pathogens and Diseases

Symposium organized by Elke Genersch/Eva Forsgren


Demonstration of Temperate Bacteriophage as a Lytical Agent of Paenibacillus larvae subsp. larvae Culture in MYPGP Medium

Jaroslav Hrabák 1, Oldřich Benada 2, Olga Hrušková-Heidingsfeldová 3

1) Department of Microbiology, Faculty of Medicine in Plzeň, Charles University, Plzeň, Czech Republic;

2) Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic;

3) Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic;


E-mail: jaroslav.hrabak©lfp.cuni.cz

The cause of lysis of Paenibacillus larvae subsp. larvae in/on MYPGP broth/agar without sporulation was not understood. This property is important to design an appropriate sporulation medium and to study of secreted products of this bacterium. We tested lytical activity of secreted molecules of P. larvae subsp. larvae on other bacteria (Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Enterobacter cloacae and Escherichia coli) and on the same strain of Paenibacillus larvae subsp. larvae. Simultaneously, the presence of bacteriophage in lysed culture was investigated by using direct electron microscopic methods and by isolation of phage DNA. The lytical activity of cell-free lysate on other bacteria and on fresh culture of P. larvae subsp. larvae was not found, but the bacteriophage particles were detected in high concentration in lysed culture. The results of isolation of phage DNA was not significant, because the phage ghosts in the lysates predominated. The induction of temperate bacteriophage was started by the decrease of pH below 6.4 and by the accumulation of metabolites in the culture. The bacteriophage was identified as BLA or PBL1 according to morphological properties, because its DNA sequence is not known.



Nosema disease in European bees

Julia Klee and Robert J. Paxton

School of Biological Sciences, Queen?s University Belfast, UK

Email: j.klee©qub.ac.uk and r.paxton©qub.ac.uk

Currently, three species of microsporidia from the genus Nosema have been identified in European bees, namely Nosema bombi from bumble bees (Bombus spp.) and Nosema apis and Nosema ceranae from the honey bee Apis mellifera. Using molecular genetic approaches (DNA sequences of the rRNA gene), N. bombi has been positively identified from a wide range of bumble bee species (Tay et al. 2005). It was formerly considered that N. apis was host-specific to A. mellifera and that N. ceranae was a parasite of the Asiatic honey bee Apis cerana. However, we and a number of other labs have recently confirmed the presence of N. ceranae in European honey bees. We have developed molecular genetic makers to allow rapid and sensitive discrimination among these three Nosema species and have used them to document the presence of Nosema among European bees. We thereby aim to shed light on the origins and epidemiology of Nosema among European bees.

Tay WT, O\'Mahony EM, Paxton RJ (2005) Complete rRNA gene sequences reveal that the microsporidium Nosema bombi infects diverse bumblebee (Bombus spp.) hosts and contains multiple polymorphic sites. Journal of Eukaryotic Microbiology 52, 505-513.


Disease removal by altered flight behavior of forager honey bees (Apis mellifera) infested with Nosema apis

J. Kralj, S. Fuchs, J. Tautz

National institute of Biology, Slovenia, E-mail: jasna.kralj©nib.si

Varroa destructor, a major pest of honey bees, alters flight behavior of foragers to a degree that may prevent their successful return to the colony. Infested workers have impaired orientation and need more time to return to the colony. As V. destructor is a relatively recent pest of the western honey bee Apis mellifera and thus specific adaptations can not be expected, it would be possible that the behavioral response is more general in nature. Here we tested the flight behavior of foragers infested by Nosema apis. Sampling departing and returning bees at the entrance showed lower infestation of returning workers compared to departing workers indicating higher loss of infested bees. Infested workers took 1.7 times longer to return to the colony than uninfested workers when released from the same location. Prolonged flights were also confirmed by labeling workers with radio frequency identification tags (RFID) to register their departure and return at the colony entrance. This data also showed that infested workers had shorter life spans and vanished from the colony earlier. The study shows a similar effect of parasitism on flight behavior of foragers infested by N. apis to that shown for V. destructor suggesting that the altered flight behavior of foragers is a general response by diseased bees and it is not limited to infestation by V. destructor. This behavior can be interpreted as suicidal pathogen removal, serving as a disease defense mechanism which reduces the colony’s load of parasites or pathogens.


Cross infectivity and impact of Nosema bombi on colony development of Bombus terrestris

Jozef van der Steen, Jeroen. Donders

PPO Bee Unit, Wageningen University and Research Centre, the Netherlands

Email: Sjef.vandersteen©wur.nl

A Nosema bombi infection can be induced via administration of N. bombi spores to host brood. The open larval stage is the brood stage in which a specific amount of spores can be administered artificially. Cross infection studies were conducted by individual administration of 5 µl of a N. bombi spore suspension in sucrose-solution 12.5% (w/v) to larvae in open cells. The adults that emerged from these cells were diagnosed within 24 hours after emergence. Administration of N. bombi spores from both B. pascuorum (250 000 spores per larva) and B. hypnorum (500 000) did not result in a microscopically detectable infection whereas comparable numbers of N. bombi spores for B. terrestris did result in an infection. The impact of N. bombi on colony founding and transmission was studied by inducing infection with N. bombi spores from B. terrestris adults to individual worker and queen larvae in open cells of B. terrestris colonies. Administration of 312 500 spores to individual queen larvae resulted in a N. bombi infection in part (30%) of the queens. Mating and hibernation were not affected but colony founding was impeded. Molecular diagnosis (ITS-f2/r2) of N. bombi in queen fed with spores showed N. bombi in part of the queens, in both the intestines and ovaries or only in the ovaries. Transmission in a colony was studies by infecting worker larvae. During colony development age cohorts were marked: adults that emerged before spores administration, adults that were supplied with N. bombi spores in the open larval stage, adults that were in the egg stage when the spores were supplied and adults that were ovipositioned 11 days after spores administration. Infection developed in the workers treated in the larval stage and was transmitted to the future age cohorts in the colony and to the adults that were in the colony before the introduction of the N. bombi infection. The brood nests of the Nosema-infected colonies were smaller than of the non-infected colonies.




Spreading of the causative agent of European foulbrood in bee colonies before and after the sanitation of an infected apiary

A.Roetschi, H.Berthoud, A. Imdorf, R. Kuhn, J-D. Charrière

Swiss Bee Research Centre, Agroscope Liebefeld-Posieux, Schwarzenburgstrasse 161, 3003 Bern, Switzerland

European foulbrood (EFB) newly infested apiaries have strongly increased during these last years in Switzerland. Therefore it is questionable if the control measures implicating the destruction of colonies with clinical symptoms, the burning of the brood material and the disinfection of beehives are sufficiently effective to maintain the bacterium Melissococcus plutonius, the causative agent of EFB, under the threshold damage.

In order to quantify the amount of M. plutonius in bee samples, a novel real-time PCR system has been established. Testing this system with samples analysed previously with the hemi-nested PCR system developed by Djordjevic and co-workers (1998) gave results totally in accordance with the hemi-nested system, thus confirming the specificity of our system towards M. plutonius.

For this study, 14 infected apiaries have been investigated. Samples were taken before the sanitation and later, in autumn before the wintering period and the following spring. Bee samples have been collected in brood nest and hive entrance from up to 8 colonies per apiary. Following DNA extraction, the samples were subjected to real-time PCR analysis.

Results show that the amount of M. plutonius has markedly decreased after the sanitation of infected apiaries but not all colonies could be brought to the wintering period free (i.e detectable amounts) of EFB. It is also noticeable that hive entrance bees were less infected than brood nest bees.



Changes in the bee pathology chapter of the new edition of the OIE manual

Wolfgang Ritter

CVUA Freiburg Germany, E-mail: ritter©bienengesundheit.de

Due to transport of bees and bee products across borders and continents, an increasing spread of pathogenic agents was observed. Therefore, diagnostic laboratories have to update their diagnostic techniques on a regular basis. The International Animal Pest Code of the OIE denominating the status of individual countries or regions concerning the existence of bee diseases as well as the transport of bee colonies within the respective regions requires a harmonisation and standardisation of diagnostic methods. Therefore the chapters on bee diseases of the “OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals”. have to be updated from time to time.

Nosema ceranae and Nosema apis in France: co-infections in honey bee colonies

M.-P.Chauzat, M. Higes, R. Martin , A. Meana ,J.-P. Faucon

AFSSA, E-mail: mp.chauzat©afssa.fr

Nosemosis is a severe pathology caused by the microporidian Nosema spp. So far only two microsporidian parasites have been described in honey bees: Nosema apis and Nosema ceranae. N. apis has been detected in European honey bees (Apis mellifera) and was one of the earliest described microsporidians (Zander, 1909). N. ceranae has been initially described in Asian bee colonies (Apis cerana) and was recently found in A. mellifera colonies in Taiwan and Europe. Very few data on this parasite are available specially on its distribution, while Nosema apis is known to be worldwide distributed. Information on symptoms resulting from N. ceranae infections in A. cerana or A. mellifera colonies are limited. On the opposite pathology symptoms due to N. apis are well documented.

Cross infections between the two host species have demonstrated that N. apis is infective in A. cerana, although its development is less effective in the Asian host compared to the European one. Spores produced by the two Nosema species are quite similar and can hardly be distinguished by traditional light microscopy analysis. In this work PCR techniques were used in order to characterize infections or co-infections by these two pathogens in French honey bee colonies. Samples were collected in France, PCR analysis were run in Spain. A total of 46 honey bee samples were analysed. Prior PCR analysis, spores were visually detected in samples. Samples have been collected during the years 2003, 2004 and 2005. The first results have shown that N. ceranae was well distributed in France: sequences of N. ceranae were found in different places distant from hundred kilometres. Coinfection with N. apis and N. cerenae were demonstrated in 3 samples. All these results show that N. ceranae has been present in France for several years.




A new approach for American Foulbrood prevention

Elena Bessi

AAT - Advanced Analytical Technologies Ltd, E-mail. elena.bessi©unicatt.it

American Foulbrood (AFB) is the most widespread and destructive brood diseases caused by the spore forming, aerobic bacteria Paenibacillus larvae subsp. larvae. The occurrence of this disease has considerable economic impact on the bee industry, as well as valuable in terms of pollination and production of honey and beeswax. The best treatment for this disease is the destruction of hives by burning, but some Countries autorised the use of antibiotics, like oxytetracycline and tylosine, to treat infected hives. Unfortunately, sometimes these treatments were incorrectly applied to the tratment of AFB development causing spreading of antibiotic resistance and pollution of honeybees products. Lactic Acid Bacteria (LAB) represent a new valuable tool for the prevention of AFB disease. They are normal inhabitants of human and animal gut and are also been found in adult and larvae of honeybees, with particular reference to lactobacilli. Some studies demonstrated that some microbes, belonging to the genus Lactobacillus, can develop beneficial actions on gastro-intestinal diseases prevention and can contribute to maintain host’s health. We previously found that some LAB, normal inhabitants of honeybees gut, may inhibit in vitro the growth of AFB- and EFB-causing bacteria. The aim of the present study, was the investigation of the possibility to in vivo prevent AFB development in honeybees by the use of selected probiotic bacteria. We selected healthy colonies to infect artificially and made three group:

A – control (non treated);

B – treated group with probiotic administration before the infection;

C – treated group with probiotic administration immediately after the infection.

We observed the presence on combs of diseases symptoms as concave and dark capping, dead larvae or scales and monitored the presence of Paenibacillus larvae and our probiotic on larval gut by mean of microbiological counts. We observed a decrease of disease symptoms on treated hives.


The incidence of honeybee parasites and diseases in Turkey

I. Çakmak1 and L. Aydin2 see fulltext

1 Uludag Universitesi, Mustafakemalpasa MYO, M.Kemalpasa-Bursa,Turkey

2 Uludag Universitesi Veteriner Fakultesi, Parazitoloji ABD, Bursa,Turkey

E-mail of the corresponding author: icakmak©uludag.edu.tr

Parasitic mite, Varroa destructor is well known parasite of honeybee and was detected in almost all apiaries that were sampled in Turkey. However, varroa mites were not detected in some colonies. Therefore, some levels of resistance to varroa might be developed in the endemic populations over time. On the other hand, there are open debates about the existence of tracheal mites, Acarapis woodi in Turkey. Interestingly, A.woodi was not found from large no. of workers sampled in most regions of Turkey even though it was reported in most of the neighbouring countries (Ellis and Munn 2005).

Nosema (Nosema apis) and Amoeba (Malpighamoeba mellificae) disease level is usually higher in Blacksea and Marmara region more than other regions. Precipitation but not the temperature was a significant factor for predicting nosema in wet regions. No data has been reported about Nosema cerena cases in Turkey yet. Brood diseases, AFB (Paenibacillus larvae larvae) and EFB (Melissococcus plutonius) are not widespread and only very few cases were reported (Simsek & Ozcan 2001; Ozkirim & Keskin 2002). Chalkbrood (Ascophaera apis) was transmitted to Turkey by imported wax in 1986 and did spread all over Turkey.

Wax moth (Galleria mellonella) is common and becomes a major problem in some years particularly in Egean and Mediterranean regions. Braula is seldom seen and small hive beetle (Aethina tumida) has not been detected yet. There is not much known about honeybee viruses in Turkey. Bee paralysis or “hairless black syndrome” is the most common case of virus disease in Anatolian bees reported by Ruttner (1988).


First Steps Towards The In vitro Cultivation of Nosema ceranae

C. Delaguila1 , F. Izquierdo1, P.G. Palencia2, R. Martin2 , M. Higes2, S. Fenoy1

Laboratorio de Parasitología, Facultad de Farmacia, Universidad San-Pablo-CEU, Madrid, Spain a. Centro Apicola Regional, Marchamalo, Guadalajara, Spain b., E-mail: cagupue©ceu.es

Microsporidia are known to infect nearly all animal phyla, but insect microsporidia are the commonest. Although the transmission of insect microsporidia to mammals is improbable, it has recently been considered possible. Nosema ceranae has recently been recogniced as a microsporidian pathogen of the honey bee Apis mellifera. Establishment of an in vitro propagation system would enable pathobiological studies of this parasite. The production of large numbers of spores would allow the development of different types of in vitro and in vivo studies, including improvement of diagnostic methods by production of monoclonal and polyclonal antibodies.

For this purpose, we have infected Vero-E6 cell with N. ceranae spores obtained from an autochthonous natural infection. Infected cells were cultured under different conditions. Most developmental as well as spore-forming stages were observed around the nucleus. Differences in growth rate were observed related to growth temperature. Results will be discussed.

Future experiments with different cell lines and culture media are needed to improve the harvest rate of spores. However, these preliminary experiments show the capability of N. ceranae to develop in in vitro culture. Furthermore, as mammal cells were infected at 37C, this opens up the possibility of them being a source of human microsporidiosis.


HONEYBEE DISEASE CONTROL IN THE EU: THE COMMISSION\'S DECISION FOR GRANTING EXEMPTIONS TO THE PRESCRIPTION-ONLY PRINCIPLE

Franco Mutinelli, Alessandra Baggio

Istituto Zooprofilattico Sperimentale delle Venezie,35020 Legnaro (PD), Italy,


E-mail: fmutinelli©izsvenezie.it

Under the new EU veterinary pharmaceutical legislation, all veterinary medicinal products used in food-producing animals will require a medical prescription from 1 January 2007. According to the commission\'s draft decision exemptions could be granted if all of the following requirements were met: 1. the administration is restricted to formulations requiring no particular knowledge or skill in using the products; this applies in particular to formulations administered by oral or topical route; 2. if administered incorrectly, the drug is not at a concentration which might present a direct or indirect risk to the animal treated or the person administering the product; 3. there are no warnings of potential side effects from correct use of the product according to its summary of product characteristics. In particular, the veterinary medicinal product or another product with the same active substance must not have previously been the subject of extensive pharmacovigilance adverse drug reaction reporting; 4. the summary of product characteristics does not refer to contraindications related to other products commonly used without prescription; 5. the product is not subject to special storage conditions or unusual conditions for safe disposal of used/unused material, including the containers; 6. there is no unnecessary risk for consumer safety as regards unacceptable residues in food obtained from treated animals or the development of resistance to antimicrobials in case the products are used incorrectly; and 7. the product contains only active substances included in Annex II of Council Regulation (EEC) No 2377/90 on the establishment of maximum residue limits (MRLs).




An approach to Nosema ceranae control with fumagillin in field conditions

Higes Mariano1, Martín-Hernández Raquel1, Garrido-Bailón Encarnación, Meana Aranzazu2

1 Regional Apicultural Center. Junta de Comunidades de Castilla - La Mancha. Marchamalo. Guadalajara. Spain.

2 Department of Animal Health. Faculty of Veterinary. Complutense University of Madrid. Spain.
E-mail: mhiges©jccm.es

The honeybee nosemosis incidence is increasing significantly in Spain for the last years and the recent detection of Nosema ceranae could be related with this fact. Fumagillin has been shown effective in Nosema apis treatment (Katznelson and Jamieson, 1952). However, no data are available about the N.ceranae control with this antibiotic or its capacity to inhibit parasite intracellular development.

A total of 20 hives randomly collected were analyzed to the detect to pathogens presence. Bee samples came from 14 apiaries belonging to 3 professional beekeepers (with around 2.000 hives distributed in east and central Spain), who had observed a clear signs of population depletion and mortality of colonies, and a decrease on honey production like the only symptoms. A high Nosema spp spores level were observed in all the samples and the specie was confirmed as Nosema ceranae by molecular diagnosis (Higes et al., 2006).

A total of 120 mg of fumagillin (CEVA) per hive was administered, dividing the total dose in four treatments once a week. The antibiotic was dissolved in 500 ml of water/sugar solution at equal quantities (w/v). Bee samples were taken one week after the third and fourth treatment, from the same colonies and again analyzed to detect Nosema spores. Three samples were on positives after the third one (light microscope) and no one after the fourth treatment (confirmed afterwards by PCR method).

Any adverse effects were not observed after the antibiotic administration and the beekeeper notify a satisfactory colony evolution.

Effects of natural compounds on Nosema diseased honeybees in laboratory conditions

Marco Lodesani1, Lara Maistrello2, Cecilia Costa1, Francesco Leonardi2, Giovanna Marani2

1Istituto Nazionale di Apicoltura, Via di Saliceto 80, 40128 Bologna, Italy; 2Dipartimento di Scienze Agrarie, Università di Modena e Reggio Emilia, Via J. F. Kennedy 17, 42100 Reggio Emilia, Italy; E-mail of the corresponding Author: mlodesani©inapicoltura.org

Nosemosis is one of the most widespread honeybee diseases, caused by the microsporidian Nosema apis. The only effective substance against this parasite, fumagillin, is no longer available for use in most European countries. For this reason, the potential of some natural compounds for the control of Nosema infection in honeybees was investigated.

Newly emerged adult bees were artificially inoculated with a known amount of N. apis spores by force-feeding the bees with a sucrose suspension. Groups of 30 bees were kept in small cages for 25 days and were fed with candy treated with thymol, resveratrol, vetiver oil, lysozyme or with untreated candy. Two randomly chosen individuals were collected from each cage at different times and examined to measure the progress of the infection; the number of dead and living bees was recorded every 3 days. From the results it emerged that thymol and resveratrol were the most promising in effectively reducing Nosema infection.
Reliability of diagnostic methods to detect Nosema spp. spores in honey bees: molecular identification versus visual observation

Martín-Hernández Raquel1, Higes Mariano1, Garrido M. Encarnación1, García-Palencia, Pilar 2, Meana Aranzazu 2

1 Regional Apicultural Center. Junta de Comunidades de Castilla - La Mancha. Marchamalo. Spain.

2 Faculty of Veterinary. Complutense University of Madrid. Spain.
E-mail: mhiges©jccm.es

Two direct methods to detect Nosema ceranae in Apis mellifera were tested to establish the reliability of diagnosis. A total of 87 bee samples were analyzed by visual observation of Nosema spores by way of the OIE recommendations as well as by PCR and posterior sequenciation. To visualize the spores, the abdomens of ten old honey bees from each sample were macerated in 5 ml of distilled water. The suspension was filtered and a second 5 ml of water used to rinse. The suspension was centrifuged for 6 min at 800 g. The pellets were resuspended again in 1 ml. Spores were identified under phase contrast microscope (x400) in 0,1 ml of the sediment.

Molecular diagnose required previous spore germination that was induced in all the samples with the following profile: 0,5 ml of the resuspended pellet of each sample was mixed with 200 µl of freshly prepared germination buffer (0.5M sodium chloride, 0.5M sodium hydrogen carbonate, pH to 6.0 with ortophosphoric acid; De Graaf et al., 1993) and incubates at 37o C for 15 minutes allowing spores germination (Rice, 2001). DNA extraction, PCR amplification and sequencing of the 16S rRNA sequence were done as described previously (Higes et al., 2006) in all the samples. PCR positive samples were 81,6% while only in 53,3% samples spores were MO observed (sensitivity 69%; specificity 100%). Only 5 samples were identified as Nosema apis and 82 were Nosema ceranae.

Work financed by INIA RTA2005-00152 and JCCM 05-280/PA-47.


Influence of sampling in the detection of Nosema ceranae spores

Martín-Hernández Raquel1, Higes Mariano1, Garrido M. Encarnación1, Meana Aranzazu 2

1 Regional Apicultural Center. Dirección General de la Producción Agropecuaria. Consejería de Agricultura. Junta de Comunidades de Castilla - La Mancha. Marchamalo. Guadalajara. Spain.

2 Department of Animal Health. Faculty of Veterinary. Complutense University of Madrid. Spain.
E-mail: mhiges©jccm.es

An infected hive parasitized with Nosema ceranae (molecular and morphologically characterized) was analyzed by visual observation of Nosema spores by way of the OIE recommendations during spring and summer months. Bee samples were taken in the morning at 8,30 h and 12,30 h. Hive entrance was sealed for half an hour; bees arriving were collected and sample marked as exterior bees. Lately another group of bees were brushing from combs close to breeding nest, and sample marked as internal bees.

The abdomens of ten honey bees from each sample were macerated in 5 ml of distilled water. The suspension was filtered and a second 5 ml of water used to rinse. The suspension was centrifuged for 6 min at 800 g. The pellets were resuspended again in 10 ml. Spores were counted under the cover-slip of a haemocytometer. In a second trial, 30 interior and 30 exterior bees collected the same day at 12,30 h were individually analyzed using the same method.

Parasite burden measured by the number of spores per bee are clearly influence either by the type of bee as well as the time of sampling. Exterior bees presented a much higher level of infection (10 fold) than the interior bees, usually collected to diagnose nosemosis. Both interior and exterior bees had almost double quantity of spores when collected at 12,30h than at 8,30 h.

In the second trial, only 15% of interior bees were positives while 50% of the exterior ones.

It is concluded that exterior bees collected at noon are a more reliable sample in spring and summer.

Work financed by INIA RTA2005-00152 and JCCM 05-280/PA-47.

Experimental infection of Apis mellifera honeybees with Nosema ceranae spores

Higes Mariano1, Martín-Hernández Raquel1, Meana Aranzazu2, García-Palencia Pilar2

1 Regional Apicultural Center. Junta de Comunidades de Castilla - La Mancha. Marchamalo. Guadalajara. Spain.

2 Department of Animal Health. Faculty of Veterinary. Complutense University of Madrid. Spain.
E-mail: mhiges©jccm.es

In this report, we describe an experimental infection of Apis mellifera by Nosema ceranae, a newly reported microsporidium.

Newly emerged Nosema free honeybees were inoculated with 125.000 Nosema ceranae spores per bee, collected from live heavily infected bees.

Three replicates cages of 20 honeybees each were done and one control cage (n=20) was done additionally which received 10µl of plain sucrose solution.

Each cage was checked daily and any dead bees counted and removed. One bee of every group was collected 3h, 3, 6 and 7 days p.i. and ventriculus prepared for MO and ME study.

Control bees were negative all through the study. Epithelial morphology showed no alterations due to methodology. Only one bee died on day 7 p.i.

In the infected groups, mortality was not observed until day 6 p.i. (33,3% ± 5%). In the three infected replicates, 94,1% bees died on day 7 p.i.

At 3 hours p.i., ventriculi looks healthy and only mature spores were observed in the intestinal lumen. Although some spores appeared empty, infected cells were not observed at that moment.

On day 3 p.i., ventriculi appeared whitish and shrunken; epithelial cells of samples from the three replicates were infected. Only the superficial epithelial layer presented all the intracellular parasite stages of the endogenous life cycle. Infected cells filled with different parasitic stages were scarce and surrounded by healthy uninfected ones. Emptied spores were observed inside infected cells.

On day 6 p.i., all the epithelial layers were affected in three replicates. Superficial layers were loaded with more mature stage.




Effect of sanitizing and curative treatments on the infestation of bee colonies by the spores of Paenibacillus larvae subsp. larvae

Krystyna Pohorecka1, Dariusz Gerula2, Małgorzata Bieńkowska2, Beata Panasiuk2

1 National Veterinary Institute-National Research Institute.

2 Institute of Pomology and Floriculture, Apiculture Division, 24-100 Pulawy, ul. Kazimierska 2

The objective of the study was to assess the health status of all bee colonies in an apiary in which clinical symptoms of American foul brood were found and to examine the impact of different sanitizing and curative treatments on the infestation level by Paenibacillus larvae larvae of bee colonies in the subsequent beekeeping season. The study was run in the years 2004-2005 in a 26-colony strong apiary. In 2006 upon inspection 3 colonies tested positive for the American foul brood. From the diseased colonies comb sections with lesioned brood were taken for diagnostic tests and from all remaining colonies both honey samples and bee samples were taken in order to test them for the spread of the disease. As a result of microbiological tests P. l. larvae was isolated and identified in 29 bee colonies. Depending on infestation level by the spores of P.l. larvae (number of bacterial colonies isolated on growth media). The colonies were divided into groups and subjected to different disease control treatments.

Group 1 – colonies with clinical symptoms of American foul brood and colonies highly infested with P.l.larvae transferred to clean hives with foundation comb and fed an antibacterial formula administered in sugar syrup.

Group B – colonies medium-highly infested not transfered but given syrup with an antibacterial formula

Group C – low infested colonies subjected to no sanitizing or curative treatments

Group D – colonies not found positive for P.l. larvae – not subjected to any sanitizing or curative treatments

In the beekeeping season of 2005 colonies were resampled for honey and bees to be tested for the presence of P.l. larvae.

From among 10 bee colonies of group A, resettling and treatment with curative formula notwithstanding, 5 colonies died by May of 2005. Of the surviving colonies, three still tested positive for P.l. larvae but the infestation level substantially declined. Only in 2 colonies of that group no P.l. larvae was found. In the group which was subjected to curative treatment only (B) two colonies died, their infestation level rising to high. Likewise, in three out of the remaining 8 colonies of that group an infestation level by P.l. larvae higher than that in 2004 was found whereas in 5 colonies infestation stayed at the same level. In the colonies of Group C in which the infestation level was low and hence they were subjected to no treatments there were also losses amounting to 3 colonies. The laboratory tests confirmed infection by P.l.larvae in four colonies that stayed unchanged from 2004, in one of them clinical symptoms having occurred and two colonies testing negative for the presence of P.l. larvae.

In group D, free of infestation in 2004, the following season the presence of P.l. larvae was found in 2 colonies.

Based on the tests performed it can be stated that in colonies in which clinical symptoms of American foul brood occur or in which the infestation level by P.l. larvae is high not even resettlement or treatment with antibacterial drugs can yield the results expected. The presence of colonies sub-clinically infested with American foul brood enhances infestation level of the colonies as well as promotes further spread of the disease.




Identification of virulence factors of P. larvae

Ainura Ashiralieva1, Rainer Borriss2, Elke Genersch1*

1 Institute for Bee Research, Friedrich-Engels-Str. 32, 16540 Hohen Neuendorf, Germany

2 Institute for Biology, Humboldt University Berlin, Chausseestr. 117, 10115 Berlin, Germany
E-mail: elke.genersch©rz.hu-berlin.de

Paenibacillus larvae (P. larvae) is the causative agent of American foulbrood, a globally spread bacterial disease of honey bee brood. AFB is highly contagious and able to kill infected colonies. Despite its disastrous economic impact on apiculture little is known about the molecular pathogenesis of this disease. Here we report first attempts to identify virulence factors of P. larvae using comparative genomics. By serially passaging a virulent wild type strain of P. larvae on nutrient agar we obtained an attenuated laboratory strain of P. larvae. In exposure bioassays this attenuated strain revealed both, a higher LC50 and a higher LT50 than the wild type strain. Hence, its virulence was reduced. When the genomes of both strains were compared by using the method of subtractive suppression hybridization (SSH) four candidate genes presumably involved in virulence determination were identified. Further studies will characterize these genes in detail and evaluate their role in the molecular pathogenesis of P. larvae.



Spread of American Foulbrood through Aethina tumida?

Marc Schäfer

CVUA Freiburg, E-mail: marc.schaefer©cvuafr.bwl.de

American foulbrood is a serious disease of honeybeebrood. The disease is caused by the spore-forming bacterium Paenibacillus larvae. Small hive beetles (Aethina tumida) and his larvae feed on honey, pollen and beebrood. As the beetle is a very active and persistent flyer, we examined if contamination of spores of Peanibacillus larvae through Aethina tumida is possible. First results will be presented.

QUANTITATIVE METHOD FOR THE DIAGNOSIS OF HONEY-BEE NOSEMOSIS

Gabriela Chioveanu

Institute for Diagnosis and Animal Health, Bucharest, Romania, E-mail: chioveanu.gabriela©idah.ro

The incidence of nosemosis remains at high level in Romania. Infections with Nosema apis (Zander) are acquired by the uptake of spores during feeding or grooming; also clinical signs and pathological modifications of the adult honey-bees are common with other diseases. There is presented the National Surveillance Program for Nosemosis at bee colonies.

There is established a standardized procedure in order to obtain the accuracy necessary for a diagnosis, according with the principles of the quality system. The suitable laboratory protocol assures the real and representative determination of the infectious level in bees, with Nosema spores.

The method principle is to determine the protozoan spores from bees triturate and to count them with a Bürker-Türk hemocytometer.

The paper presents the principal phases and the evaluation of the performer characteristics of the method: specificity, reproducibility, precision and incertitude of measure, compulsory parameters for to validate this procedure.

Finally, it has been established 3 principal infectious levels.

Activation of Nosema apis spores by carbon dioxide

Krystyna Czekońska

Department of Pomology and Apiculture, Agricultural University, of Krakow, Poland; E-mail of the corresponding Author: kczekon©ogr.ar.krakow.pl

Carbon dioxide (CO2) can be used to disinfect honeycombs against the most harmful pest of the honeycombs, the greater wax moth (Galleria mellonella L.). However, it is not known, how this gas affects Nosema apis which are one of the most common parasites on the honeybee. The aim of this study was to examine if treatment of N. apis spores with CO2 effects their ability to infect honeybee workers.

An inoculating solution was prepared and divided into 4 doses. Three doses were exposed to the effect of CO2 (100% concentration), for 30, 35 and 40 hours. The fourth dose was used as a control, without exposure to CO2. Each of the four doses was used to individually inoculate 120 workers from each group. The fifth group was formed of workers that were not inoculated with spores of N. apis. The cages with the bee workers were kept in an incubator, under 30ºC. The course of invasion by parasites was tested every third day, by counting the number of N. apis spores in the digestive tracts of living workers.

The significant differences in the rate of infection were observed from the 9th day after inoculation. Between groups there were significant differences in the survival rates of workers. Treatment of the N. apis spores with CO2 resulted in a faster proliferation of the parasite and higher mortality among the workers.


Molecular screening by PCR of honey bee larvae for Melisococcus plutonius infection

Pietro Arculeo, Anna Maria Di Noto, Fabrizio Vitale, , Maria Vitale.

Istituto Zooprofilattico Sperimentale della Sicilia , Palermo Italy, E-mail: arculeo©pa.izs.it

European foulbrood is a disease of honey bee larvae caused by Melisococcus plutonius and secondary bacterial invaders. The diagnosis is usually performed through bacteria isolation which can be hampered by particularly stringent growth conditions requirements and competition with other microbes.

PCR assay can be an useful, high sensitive and specific method to assays the honeybee larvae. We analysed several honey bee larvae and few isolated bacterial strains, by an amplification of a DNA fragment of 16S ribosomal RNA gene. The original protocol of Govan et al. had been modified to obtain a higher sensitivity. All bacterial strains isolated by infected honey bee larvae resulted positive, while several other bacterial DNA resulted negative.



Purification and biochemical Characterization of proteolytic enzyme of bacterium Paenibacillus larvae subsp. larvae

J. Hrabák1 , O. Hrušková-Heidingsfeldová 2, K. Martínek 1

1 Department of Microbiology, Faculty of Medicine in Plzen, Charles University, Plzen, 2Institute of Organic Chemistry and Biochemistry, Academy of Science of the Czech Republic, Prague

Introduction: Bacterium Paenibacillus larvae subsp. larvae is the most dangerous pathogen of bees due to an extremely resistance of its spores. It secretes several proteolytic enzymes during its growth, but their role in pathological processes as well as their biochemical characterization are still unclear. The results of biochemical research of these enzymes might explain their role in the pathogenesis, but it is necessary to use a purified material.

Methods: For production of proteolytic enzymes of bacterium Paenibacillus larvae subsp. larvae, The MYPGP medium was used. The basic biochemical information about these secreted enzymes were obtained by zymography. The optimal incubation conditions and inhibition pattern were detected by the same method. Inhibitors of all classes of proteolytic enzymes were used (Pepstatin, Leupeptin, E64, Iodoacetamide, PMSF, EDTA, EGTA a 1,10-Phenantroline). Gel filtration (Sephadex G-200) and ion exchange chromatography (DEAE cellulose) were proposed to purification of these enzymes.
Results: In the cell free supernatant, the proteolytic enzymes with approximately molecular weight of 87, 74, 40 and 42 kDa were detected. All of these enzymes were inhibited by inhibitors of metaloproteases, but not by inhibitors of other classes. pH optimum appeared about pH 7.0 with some differences between 87/74 kDa and 40/42 kDa enzymes. Proteolytic enzymes may be optimally purified after 24 hrs of incubation at 4°C by gel filtration followed ion exchange chromatography. Detected enzymes probably have character of exoproteases according to substrate specificity analyses.
Conclusion: We have found that the bacterium Paenibacillus larvae subsp. larvae secrets two different classes of proteolytic enzymes which probably are metalloproteases. Their exopeptidase character indicates their role in nutrition of this bacterium. Suggested purification procedure may be used to obtain of purified enzymes.


Survival of American foulbrood pathogen in hot beeswax

M. Haklová, D. Titěra, M. Lutzová, P. Janoušová, M. Bednář

Bee Research Institute at Dol, E-mail: beedol©beedol.cz

The most significant problems at the American foulbrood (AFB) control are due to the resistance of the AFB pathogen, the sporulating microbe Paenibacillus larvae. We studied the possibility of the beeswax disinfection. The experiments were based on the beeswax analysed as P.l. free. Into this beeswax there were inserted quantitatively P.l. spores (stem 643/04) obtained from the scales of the AFB combs in the quantity 9.107 CFU/g beeswax.

For the thermal exposition oil bath was assembled with submergible heater and oil air mixer. The bath temperation was controlled by calibrated thermocouple and the achieved temperature oscillation in the whole bath volume was less than 0,05 °C Elected temperatures: 90-108-121-134 °C, applied time expositions: 0,3-1-2-4-6-12-24 hours

After the thermal expositions the wax was taken into warm physiological solution and after thorough agitation and cooling the agar plates were inoculated and subsequently cultivated . The growing colonies of P.l.l. were re-counted per 1 g wax after five days.

The spores of AFB pathogen are very resistant to temperatures in the standard processing of beeswax. As to achieve safe reduction of the viable spores of P.l. more hours heat of beeswax at the temperature more than 130 °C is necessary. But the long time heat of wax changes also its chemical characteristics.

Supported by grant project NAZV 1G46032.


The use of Commercial Nutrient Agar TRIOS for diagnosis of American foulbrood in Honeybees

M. Haklová,1, E. Bazgerová2, D. Titěra1, J. Bzdil3

1 Bee Research Institute at Dol, E-mail: beedol©beedol.cz
2
Trios s.r.o., Zakouřilova 142, 149 00 Praha 4
3 SVÚ Olomouc

In the programs of the American foulbrood control there is very important in time exact diagnosis of the incidence of sporulating microorganism Paenibacillus larvae. Samples for the diagnostics are taken from various material (brood, combs, debris, honey). After the sample preparation they are cultivated on agar plates. In the accredited laboratories in the Czech Republic the cultivation is carried out on MYPGP agar prepared by laboratories by themselves. Ring test has shown that one of the variability sources is the nutrient agar prepared in small charges in individual laboratories.

On the basis of cultivating medium developed in the Bee Research Institute Dol the commercially prepared medium MKM™ MYPPN was introduced into serial production.

Validation trials have shown that the medium is suitable for routine laboratory diagnostics.

The use of the commercial medium MKM™ MYPPN is for small laboratories more cost effective than laboratory preparation of this nutrient medium.

Supported by grant project NAZV 1G46032.



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