Projects / Programmes source: ARIS

Genotyping of the American foulbrood causative agent as a tool to determine the transmission routes of the disease

Research activity

Code Science Field Subfield
4.04.02  Biotechnical sciences  Veterinarian medicine  Animal pathology and epizootiology 

Code Science Field
B230  Biomedical sciences  Microbiology, bacteriology, virology, mycology 

Code Science Field
4.03  Agricultural and Veterinary Sciences  Veterinary science 
American foulbrood (AFB), Paenibacillus larvae, transmission routes, confined zone, genotyping, ERIC-PCR, whole-genome sequencing (WGS)
Evaluation (rules)
source: COBISS
Researchers (19)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  28448  PhD Jana Avberšek  Veterinarian medicine  Researcher  2018 - 2020  121 
2.  30378  PhD Majda Golob  Veterinarian medicine  Researcher  2018 - 2020  185 
3.  25514  PhD Biljana Hacin  Biotechnology  Technical associate  2018 - 2020  27 
4.  04272  PhD Vlasta Jenčič  Veterinarian medicine  Researcher  2018 - 2020  436 
5.  11236  Mira Jenko Rogelj  Veterinarian medicine  Technical associate  2018 - 2020  65 
6.  39657  Maja Kavalič    Technical associate  2018 - 2020 
7.  38142  PhD Tanja Knific  Veterinarian medicine  Researcher  2020  73 
8.  18104  Olga Kosar  Veterinarian medicine  Technical associate  2018 - 2020 
9.  24296  PhD Darja Kušar  Veterinarian medicine  Head  2018 - 2020  201 
10.  17874  Maja Lepen  Veterinarian medicine  Technical associate  2018 - 2020 
11.  11133  PhD Matjaž Ocepek  Veterinarian medicine  Researcher  2018 - 2020  469 
12.  38144  PhD Bojan Papić  Veterinarian medicine  Researcher  2018 - 2020  108 
13.  24612  PhD Mateja Pate  Veterinarian medicine  Researcher  2018 - 2019  280 
14.  15315  PhD Metka Pislak Ocepek  Veterinarian medicine  Researcher  2018 - 2020  152 
15.  37058  Jerica Vreček Šulgaj  Veterinarian medicine  Technical associate  2018 - 2020 
16.  33447  Urška Zajc  Veterinarian medicine  Technical associate  2018 - 2020  79 
17.  12682  PhD Irena Zdovc  Veterinarian medicine  Researcher  2018 - 2020  465 
18.  50724  Alenka Žugelj    Technical associate  2018 - 2020 
19.  26499  Lucija Žvokelj  Animal production  Technical associate  2018 - 2020  52 
Organisations (1)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0406  University of Ljubljana, Veterinary Faculty  Ljubljana  1627139  10,745 
Beekeeping has a long tradition in Slovenia. It is an important agricultural and economical branch on one side, while on the other it plays a unique role in preserving the national cultural heritage. The main purpose of the beekeeping is still the production of bee products (honey, pollen, propolis, wax, royal jelly and bee venom), which are indispensable in the human consumption and have evident positive effects on human the health; in addition, they are also used in the pharmaceutical industry. Furthermore, the production of queens and manufacture of the beekeeping equipment are also an important part. Bees play a very important role by pollinating cultural plants and, therefore, have an invaluable economic and nature-conservation meaning in a broad sense. A more and more increasing activity in Slovenia, linked to the unique beekeeping heritage and culture, is the beekeeping tourism.   In Slovenia, beekeeping is allowed only with the Slovene autochthonous bee race Apis mellifera carnica (the Carniolan grey bee), which has characteristic favourable features, both morphological and ethological. There are queen breeding apiaries in Slovenia, which are used to breed the indigenous Carniolan bee. An important beekeeping goal is also increasing the resistance of the bees against diseases. Bee societies are subjected to several contagious diseases, among which the varrosis and the American foulbrood are the most important from the economical perspective.   The American foulbrood (AFB) is an extremely contagious and one of the most devastating diseases of the bee societies. It occurs mostly in the areas populated by the honey bee A. melifera. The causative agent of the diseases is a Gram positive sporogenic bacterium Paenibacillus larvae, which affects very young bee larvae. The adult bees are not susceptible to P. larvae infection. The larvae get infected with P. larvae spores through the contaminated food. The spores germinate in the intestinal lumen of larvae, bacteria multiply to a great extent, invade the haemolymph and cause the death of larvae. The latter is then decomposed into a sticky mass, which later becomes a dry scale containing millions of spores. Spores of P. larvae are extremely resistant against several environmental factors and may remain infective for decades. Because of the disease, a severe deficit of the bee brood appears; the diseased bee societies weaken and are completely destroyed.   P. larvae infection may be latent for months; meanwhile, the causative agent P. larvae spreads unimpeded within the bee society. The most efficient measure to suppress the disease is to prevent its spread. In accordance to the Regulation on the measures to detect, suppress, inform and prevent American foulbrood, the disease should not be treated by drugs and should be notified to the authorities according to law. Suppression of the disease includes harmless destruction of the infected bee societies, contaminated beehives and infected combs, in addition to the equipment that cannot be disinfected. On rare occasions, in the bee societies without clinical signs and in the strong bee societies where the disease is still in an initial stage, the use of the bee shaking method is allowed. Since the disease is often spread by the uncontrolled bee transportation, one of the suppression measures in Slovenia is the introduction of 3-km zone around the disease focus (so called infection circle or embargo zone). In this zone, the transport of bees is prohibited until the focus is sanitized and all the bee societies within the zone are inspected. If there is a new focus detected in the zone, the transport ban is expanded accordingly. This may take several months and cause additional economic losses due to the ban on the transport of the bees to the honey flow areas and the ban on the trade with queens and bee societies. There are differences between the states regarding regulations for the suppression of American foulbrood, particularly i
Significance for science
Whole genome sequencing (WGS) is currently the ultimate method for typing of bacterial isolates, which has been rarely used for the purpose of typing P. larvae despite the importance of this pathogen. The project will greatly expand the use of WGS for the epidemiological surveillance of American foulbrood on a global scale and provide an insight into the genetic diversity of epidemiologically related P. larvae isolates. Furthermore, WGS will define the outbreaks of American foulbrood in more detail. The results will improve the knowledge on the disease transmission routes and examine the effectiveness of the current disease control guidelines, including the size of the inspection radius. We will establish a national database of the whole genome sequences of P. larvae, which will serve as a basis for the surveillance of the disease in Slovenia. All the obtained whole genome sequences will be deposited in the publicly available NCBI SRA database and will thus be accessible to researchers around the world. The whole genome sequences of P. larvae will also be compared to other, previously published whole genome sequences of P. larvae that originate from different geographical areas. Moreover, for all the sequenced isolates, we will also determine their corresponding MLST sequence types, which will give us insight into the population structure of P. larvae in Slovenia and put it on the global map.   All P. larvae isolates that will be analyzed within the framework of the project will be typed using ERIC-PCR. This will determine the prevalence of different ERIC types in Slovenia in conjunction with the associated clinical picture of the disease. Based on the obtained results, we will propose potential changes to the disease control guidelines, depending on the genotype of the pathogen. The implementation of ERIC-PCR will allow the routine typing of P. larvae isolates (e.g. in prospective outbreak investigations).   Within the framework of the project, we will implement molecular methods (qPCR and dPCR) for the quantification of P. larvae in honey samples, which will be an important contribution in the field of molecular quantification of this pathogen. Based on the number of spores in honey and the clinical condition of the corresponding apiary, we will determine the predictive value of the number of spores in honey as an indicator for the possible occurrence of the disease.   The results that will be obtained within this project will be presented to the national and international public at professional and scientific meetings. The project members plan to publish the results in at least two scientific articles in journals with an impact factor. The work that will be performed within the project will also contribute to the international exchange of knowledge and the promotion of science in Slovenia. New insights will be incorporated into beekeeping practice as well as educational and research work at the Veterinary Faculty, University of Ljubljana.
Significance for the country
The aim of the project is to study the modes of transmission of American foulbrood and to improve the preventive and eradication measures in order to decrease the occurrence of the disease. In the case of disease outbreak, the ban on the transport of honeybees and equipment within the radius of 3 km from the outbreak focus is established; we aim to evaluate whether this measure is appropriate for sanitation of the affected area or the ban area could be diminished. Altogether, this could mean less disease foci, less affected and destroyed bee societies and diminished ban area where trade and transport are forbidden. The ban on bee transport within the 3-km zone affects all beekeepers in that area, including the beekeepers whose bee societies are healthy. This measure particularly affects the professional beekeepers. Based on the results of the project, the state could reduce the costs for the compensations to the beekeepers to which they are entitled due to the destruction of bee societies and all beekeeping equipment when they report the disease suspicion by themselves. The costs of clinical examinations of the honeybee societies in the apiary and laboratory investigations could also be reduced.   In the case of American foulbrood outbreak and consequently the destruction of bee societies, an even bigger economic loss is caused by the loss of bee products such as honey, pollen, propolis, wax, royal jelly and bee venom which are indispensable in healthy food, either per se or processed into different food products; they are also used in the pharmaceutical industry and in beekeeping. Production and processing of the bee products may be a successful supplementary activity on farms. When bee transportation is limited due to a ban, the bee transport to the honey flow areas is prohibited, leading to a decreased yield of the monofloral honey of higher quality schemes and honey of geographic origin. In the ban area, it is also prohibited to keep, sell and export the queens of Slovene indigenous bee race Apis mellifera carnica, which is protected by the law. Queens of A. mellifera carnica, the bee race also called the Carniolan gray bee, are due to their good characteristics highly demanded abroad and the trade ban represents a loss of competitive place on the foreign markets.   Bee colony losses due to American foulbrood and the ban on transport to the honey flow areas reflect also in a decreased pollination activity of bees, which is of invaluable economic and nature preserving importance. The fruit harvest, which is a result of bee pollination, is not only richer but also of a higher quality. Some data suggest that one third of the consumed food depends on the existence of bees. Food production is gaining importance due to the population increase and global warming. Indirect pollination of plants and consequently a richer harvest enable the development of other agricultural and industrial branches. In addition to the pollination of cultural plants, the bees also play an important role in nature preservation as they pollinate the insect-pollinated plants (wild plants), which are a part of natural flora of a respective area, and therefore maintain the plant biodiversity and natural balance of the whole habitat.   Slovenia is a leading country in the development of honeybee tourism which is getting more and more established as a part of a touristic offer in our country; therefore, the ban due to the disease outbreak limits also the tourism industry. Last, but not least, Slovenia gained the attention of the world bee keeping public due to the proposal to celebrate the world bee day; therefore, the control of American foulbrood should be well regulated to retain the reputation of the country of good beekeepers.
Most important scientific results Final report
Most important socioeconomically and culturally relevant results Final report
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