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Genetic diversity among isolates of Paenibacillus larvae from Austria
Journal of Invertebrate Pathology 100 (2009) 44–46
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa
Short Communication
Genetic diversity among isolates of Paenibacillus larvae from Austria Igor Loncaric, Irmgard Derakhshifar *, Josua T. Oberlerchner, Hemma Köglberger, Rudolf Moosbeckhofer Institute for Apiculture, Austrian Agency for Health and Food Safety (AGES), Spargelfeldstrasse 191, 1226 Vienna, Austria
a r t i c l e
i n f o
Article history: Received 14 March 2008 Accepted 6 September 2008 Available online 14 September 2008 Keywords: American foulbrood Paenibacillus larvae rep-PCR BOX-PCR ERIC-PCR Honeybee Apis mellifera Genotype
a b s t r a c t Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and ab). Genotypes aB and ab are new and have not been reported in other studies using the same techniques. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction The etiological agent of American foulbrood (AFB) Paenibacillus larvae is reported to cause this disease in honey bee larvae worldwide (de Graaf et al., 2006). AFB can be spread by swarms, drifting or robbing bees and beekeepers when they exchange hive equipment, combs, honey or pollen stores between colonies or apiaries. Spread of AFB spores is also reported for wax moths (Ritter, 1996). Until present, several studies have been carried out to study genetic diversity of P. larvae. For these purposes the techniques most frequently applied were ERIC-, REP- and BOX-PCR. Alippi and Aguilar (1998) used BOX-PCR to study genetically diversity of 99 strains of former P. larvae subsp. larvae, originating from different regions of Argentina and other countries. Isolates from Argentina generated three groups of patterns (designated A, B and C), while P. larvae subsp. larvae strains obtained from other countries yielded two distinguishable patterns (coincident with A and B). Using rep-PCR technique with BOX A1R and MBO REP1 primers, Genersch and Otten (2003) examined 105 German P. larvae isolates and detected four genetic subgroups (AB, Ab, ab and aB). Genersch et al. (2006) identified four different genotypes (ERIC I–IV) using ERIC-PCR. Banding patterns obtained from former P. larvae subsp. larvae were called ERIC I and II and former P. larvae subsp. pulvifaciens ERIC III and IV. Peters et al. (2006) applied the same techniques and protocols as described by Genersch and Otten (2003) for genotyping of 176 isolates originating from broodcombs and honey * Corresponding author. Fax: +43 50 555 33 133. E-mail address: [email protected] (I. Derakhshifar). 0022-2011/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.09.003
samples of 54 apiaries in the administrative district of Arnsberg (North Rhine-Westphalia, Germany). The authors detected five different genotypes (AB, Ab, ab, ab and A<). American foulbrood is a well-known notifiable disease in Austria. Until present there have not been done any studies on the occurrence and distribution of P. larvae genotypes in Austria. Therefore, the aim of this study was to genotype P. larvae strains from all Austrian federal provinces. 2. Material and methods Two hundred and fourteen P. larvae strains were cultured from brood, showing clinical symptoms and having been tested positive for AFB, as well as from spore contaminated samples of extracted honey and honey supplies on brood combs. Type strain (P. larvae DSM 7030T) and several P. larvae reference strains (DSM 16115, DSM 16116, DSM 17236, DSM 3615, LMG 14428 and LMG 16252) were also included in this study. Strains were cultivated and identified according to Bakonyi et al. (2003). For rep-PCR experiments, DNA was extracted as described by Loncaric et al. (2008) with the following slight modification: after adding MD1 solution, the cells were heated for a prolonged step of 25 min. DNA was amplified by using ERIC1R-ERIC2, BOX A1R and MBO REP1 primers (Versalovic et al., 1994). Amplification reactions were performed in a GeneAmp PCR System 9700 Thermocycler (Applied Biosystem) in a 15 ll reaction volume containing 7.5 ll of a REDTaq ReadyMix PCR Reaction Mix (Sigma, Vienna, Austria)
I. Loncaric et al. / Journal of Invertebrate Pathology 100 (2009) 44–46
with 0.45 U of Taq DNA polymerase, 5–10 ng of template DNA and 1.35 of each ERIC primer or 3 lmol l 1 of BOX A1R and MBO REP1. Thermal cycling parameters were: 95 °C for 5 min, 35 cycles of 94 °C for 1 min, 59 °C (MBO), 53 °C (BOX), 55 °C (ERIC) for 1 min, 72 °C for 1.5 min (MBO) and 2.5 min (BOX, ERIC); and final extension of 72 °C for 10 min. PCR products were analyzed by electrophoresis in 1.5% agarose gel in TAE buffer run at 5 V cm 1 with 100 bp ladder (ROTH, Karlsruhe, Germany) and detected by staining with ethidium bromide (0.5 lg ml 1) under UV light and pho-
45
tographed. Differences in banding pattern between the strains were named according to nomenclature of Genersch and Otten (2003), Peters et al. (2006) and Genersch et al. (2006). 3. Results and discussion All ERIC, BOX and REP primer sets generated multiple distinct DNA fragments and shared several major bands with type strain and reference strains, which indicates a high degree of relatedness
Fig. 1. (a and b) Electrophoretic profiles of selected isolates obtained by BOX- and MBO REP primers.
46
I. Loncaric et al. / Journal of Invertebrate Pathology 100 (2009) 44–46
Table 1 Occurrence of P. larvae genotypes in Austria Federal province
Genotypes (numbers of samples)
Sum
Ab
ab
ab
AB
aB
Burgenland Carinthia Lower Austria Upper Austria Salzburg Styria Tyrol Vorarlberg Vienna
0 1 4 5 7 10 1 3 2
5 3 15 18 3 17 3 6 19
2 0 0 0 0 0 0 0 0
0 0 0 5 0 0 8 1 2
1 2 1 35 2 0 8 21 4
8 6 20 63 12 27 20 31 27
Total
33
89
2
16
74
214
Total%
15
42
1
7
35
ERIC I
ERIC II
%
58
42
The findings of the present study could be helpful in trainings for veterinarians, bee inspector’s extension staff and beekeepers, thus improving the detection of AFB infections in the field. Taking into account that the genotype has an influence on heat tolerance of P. larvae (Forsgren et al., 2007) the detection of the pathogen in honey samples could also be improved in the laboratory. Considering these results and their implications for practical extension and laboratory detection services, genotyping of isolates according to a sampling plan could be recommended. Thus, it would be possible to keep on track with possible changes of occurring AFB genotypes in a region as well as with the intrusion of new genotypes. Reliable data on these factors are necessary for efficient detection and management of American foulbrood to keep the economic damage for beekeepers as low as possible. With respect to these facts the detection of two new genotypes in Austria should be the starting point for further investigation of their characteristics on the level of the pathogen as well as the bee colony. Acknowledgments
and confirms the affiliation of the isolates as members of P. larvae. The tested Austrian isolates clustered in genotypes ERIC I (58%) and ERIC II (42%). Genomic fingerprints obtained after BOX-PCR exhibited genotypes A, a and a (Fig. 1). MBO REP 1 primer generated two banding patterns b and B (Fig. 1). Combining BOX A1R and MBO REP1 primers five genotypes were identified: ab (42%), aB (35%), Ab (15%), AB (7%) and ab (1%). Genotypes aB and ab were new and have not been reported in other studies using the same techniques. Whereas genotype aB was isolated from brood, extracted honey and honey supplies on brood combs, ab was detected only in extracted honey from one beekeeping operation with several bee yards near the Hungarian border. The occurrence of the different genotypes in the federal provinces of Austria is given in Table 1. Knowledge about the P. larvae genotypes in a country could be important for AFB risk assessment, because there is a correlation between genotype and occurrence of clinical symptoms. Genersch et al. (2005) demonstrated in exposure bioassays that genotype AB killed infected larvae faster than other genotypes. Because most larvae infected with genotype AB were dieing before cell capping, it could be hypothesized that nursing bees could detect and remove them, thus reducing the production of spores. Spreading of this genotype within the colony would therefore be slower and clinical AFB diagnosis would be handicapped because a spotty brood nest is dominating and fewer cells with perforated, sunken cappings, containing the ropy stage or foulbrood scales could be found. Consequently, unrecognized spreading of genotype AB in the field would be favored by beekeepers if they split colonies to increase hive numbers. In this study in two cases two different genotypes (ab and Ab) could be identified from the same comb of a colony. These combs came from two different beekeeping operations from two different federal provinces of Austria. A similar case was reported by Peters et al. (2006) for German P. larvae isolates, where two genotypes had been found in one apiary. Such multi-genotype infections of colonies or apiaries may increase the complexity of P. larvae infections by influencing type and speed of development of clinical symptoms.
Financial support was granted by the Federal Ministry of Agriculture, Forestry, Environment and Water Management according to Reg. (EC) No 797/2004 and the Austrian Agency for Health and Food Safety Ltd. (AGES). We thank ‘‘Biene Österreich” for support and Katharina Etter for technical assistance. References Alippi, A.M., Aguilar, O.M., 1998. Characterization of isolates of Paenibacillus larvae subsp. larvae from diverse geographical origin by the polymerase chain reaction and BOX primers. J. Invertebr. Pathol. 72, 21–27. Bakonyi, T., Derakhshifar, I., Grabensteiner, E., Nowotny, N., 2003. Development and evaluation of PCR assays for the detection of Paenibacillus larvae in honey samples: comparison with isolation and biochemical characterization. Appl. Environ. Microbiol. 69, 1504–1510. de Graaf, D.C., Alippi, A.M., Brown, M., Evans, J.D., Feldlaufer, M., Gregorc, A., Hornitzky, M., Pernal, S.F., Schuch, D.M., Titera, D., Tomkies, V., Ritter, W., 2006. Diagnosis of American foulbrood in honey bees: a synthesis and proposed analytical protocols. Lett. Appl. Microbiol. 43, 583–590. Forsgren, E., Stevanovic, J., Fries, I., 2007. Variability in germination and in temperature and storage resistance among Paenibacillus larvae genotypes. Vet. Microbiol. 129, 342–349. Genersch, E., Otten, C., 2003. The use of repetitive element PCR fingerprinting (rep PCR) for genetic subtyping of German field isolates of Paenibacillus larvae larvae. Apidologie 3 (2003), 195–206. Genersch, E., Ashiralieva, A., Fries, I., 2005. Strain- and genotype-specific differences in virulence of Paenibacillus larvae subsp. larvae, a bacterial pathogen causing American foulbrood disease in honey bees. Appl. Environ. Microbiol. 71, 7551– 7555. Genersch, E., Forsgren, E., Pentikäinen, J., Ashiralieva, A., Rauch, S., Kilwinski, J., Fries, I., 2006. Reclassification of Paenibacillus larvae subsp. Pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation. Int. J. Syst. Evol. Microbiol. 56, 501–511. Loncaric, I., Donat, C., Antlinger, B., Oberlerchner, J.T., Heissenberger, B., Moosbeckhofer, R., 2008. Strain-specific detection of two Aureobasidium pullulans strains, fungal biocontrol agents of fire blight by new, developed multiplex-PCR. J. Appl. Microbiol. 104, 1433–1441. Peters, M., Kilwinski, J., Beringhoff, A., Reckling, D., Genersch, E., 2006. American foulbrood of the honey bee: occurrence and distribution of different genotypes of Paenibacillus larvae in the administrative district of Arnsberg (North RhineWestphalia). J. Vet. Med. B Infect. Dis. Vet. Public Health 53, 100–104. Ritter, W., 1996. Diagnostik und Bekämpfung der Bienenkrankheiten. Gustav Fischer Verlag, Jena. Versalovic, J., Schneider, M., de Bruijn, F.J., Lupski, J.R., 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol. Cell. Biol. 5, 25–40.
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/yjipa
Short Communication
Genetic diversity among isolates of Paenibacillus larvae from Austria Igor Loncaric, Irmgard Derakhshifar *, Josua T. Oberlerchner, Hemma Köglberger, Rudolf Moosbeckhofer Institute for Apiculture, Austrian Agency for Health and Food Safety (AGES), Spargelfeldstrasse 191, 1226 Vienna, Austria
a r t i c l e
i n f o
Article history: Received 14 March 2008 Accepted 6 September 2008 Available online 14 September 2008 Keywords: American foulbrood Paenibacillus larvae rep-PCR BOX-PCR ERIC-PCR Honeybee Apis mellifera Genotype
a b s t r a c t Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and ab). Genotypes aB and ab are new and have not been reported in other studies using the same techniques. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction The etiological agent of American foulbrood (AFB) Paenibacillus larvae is reported to cause this disease in honey bee larvae worldwide (de Graaf et al., 2006). AFB can be spread by swarms, drifting or robbing bees and beekeepers when they exchange hive equipment, combs, honey or pollen stores between colonies or apiaries. Spread of AFB spores is also reported for wax moths (Ritter, 1996). Until present, several studies have been carried out to study genetic diversity of P. larvae. For these purposes the techniques most frequently applied were ERIC-, REP- and BOX-PCR. Alippi and Aguilar (1998) used BOX-PCR to study genetically diversity of 99 strains of former P. larvae subsp. larvae, originating from different regions of Argentina and other countries. Isolates from Argentina generated three groups of patterns (designated A, B and C), while P. larvae subsp. larvae strains obtained from other countries yielded two distinguishable patterns (coincident with A and B). Using rep-PCR technique with BOX A1R and MBO REP1 primers, Genersch and Otten (2003) examined 105 German P. larvae isolates and detected four genetic subgroups (AB, Ab, ab and aB). Genersch et al. (2006) identified four different genotypes (ERIC I–IV) using ERIC-PCR. Banding patterns obtained from former P. larvae subsp. larvae were called ERIC I and II and former P. larvae subsp. pulvifaciens ERIC III and IV. Peters et al. (2006) applied the same techniques and protocols as described by Genersch and Otten (2003) for genotyping of 176 isolates originating from broodcombs and honey * Corresponding author. Fax: +43 50 555 33 133. E-mail address: [email protected] (I. Derakhshifar). 0022-2011/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2008.09.003
samples of 54 apiaries in the administrative district of Arnsberg (North Rhine-Westphalia, Germany). The authors detected five different genotypes (AB, Ab, ab, ab and A<). American foulbrood is a well-known notifiable disease in Austria. Until present there have not been done any studies on the occurrence and distribution of P. larvae genotypes in Austria. Therefore, the aim of this study was to genotype P. larvae strains from all Austrian federal provinces. 2. Material and methods Two hundred and fourteen P. larvae strains were cultured from brood, showing clinical symptoms and having been tested positive for AFB, as well as from spore contaminated samples of extracted honey and honey supplies on brood combs. Type strain (P. larvae DSM 7030T) and several P. larvae reference strains (DSM 16115, DSM 16116, DSM 17236, DSM 3615, LMG 14428 and LMG 16252) were also included in this study. Strains were cultivated and identified according to Bakonyi et al. (2003). For rep-PCR experiments, DNA was extracted as described by Loncaric et al. (2008) with the following slight modification: after adding MD1 solution, the cells were heated for a prolonged step of 25 min. DNA was amplified by using ERIC1R-ERIC2, BOX A1R and MBO REP1 primers (Versalovic et al., 1994). Amplification reactions were performed in a GeneAmp PCR System 9700 Thermocycler (Applied Biosystem) in a 15 ll reaction volume containing 7.5 ll of a REDTaq ReadyMix PCR Reaction Mix (Sigma, Vienna, Austria)
I. Loncaric et al. / Journal of Invertebrate Pathology 100 (2009) 44–46
with 0.45 U of Taq DNA polymerase, 5–10 ng of template DNA and 1.35 of each ERIC primer or 3 lmol l 1 of BOX A1R and MBO REP1. Thermal cycling parameters were: 95 °C for 5 min, 35 cycles of 94 °C for 1 min, 59 °C (MBO), 53 °C (BOX), 55 °C (ERIC) for 1 min, 72 °C for 1.5 min (MBO) and 2.5 min (BOX, ERIC); and final extension of 72 °C for 10 min. PCR products were analyzed by electrophoresis in 1.5% agarose gel in TAE buffer run at 5 V cm 1 with 100 bp ladder (ROTH, Karlsruhe, Germany) and detected by staining with ethidium bromide (0.5 lg ml 1) under UV light and pho-
45
tographed. Differences in banding pattern between the strains were named according to nomenclature of Genersch and Otten (2003), Peters et al. (2006) and Genersch et al. (2006). 3. Results and discussion All ERIC, BOX and REP primer sets generated multiple distinct DNA fragments and shared several major bands with type strain and reference strains, which indicates a high degree of relatedness
Fig. 1. (a and b) Electrophoretic profiles of selected isolates obtained by BOX- and MBO REP primers.
46
I. Loncaric et al. / Journal of Invertebrate Pathology 100 (2009) 44–46
Table 1 Occurrence of P. larvae genotypes in Austria Federal province
Genotypes (numbers of samples)
Sum
Ab
ab
ab
AB
aB
Burgenland Carinthia Lower Austria Upper Austria Salzburg Styria Tyrol Vorarlberg Vienna
0 1 4 5 7 10 1 3 2
5 3 15 18 3 17 3 6 19
2 0 0 0 0 0 0 0 0
0 0 0 5 0 0 8 1 2
1 2 1 35 2 0 8 21 4
8 6 20 63 12 27 20 31 27
Total
33
89
2
16
74
214
Total%
15
42
1
7
35
ERIC I
ERIC II
%
58
42
The findings of the present study could be helpful in trainings for veterinarians, bee inspector’s extension staff and beekeepers, thus improving the detection of AFB infections in the field. Taking into account that the genotype has an influence on heat tolerance of P. larvae (Forsgren et al., 2007) the detection of the pathogen in honey samples could also be improved in the laboratory. Considering these results and their implications for practical extension and laboratory detection services, genotyping of isolates according to a sampling plan could be recommended. Thus, it would be possible to keep on track with possible changes of occurring AFB genotypes in a region as well as with the intrusion of new genotypes. Reliable data on these factors are necessary for efficient detection and management of American foulbrood to keep the economic damage for beekeepers as low as possible. With respect to these facts the detection of two new genotypes in Austria should be the starting point for further investigation of their characteristics on the level of the pathogen as well as the bee colony. Acknowledgments
and confirms the affiliation of the isolates as members of P. larvae. The tested Austrian isolates clustered in genotypes ERIC I (58%) and ERIC II (42%). Genomic fingerprints obtained after BOX-PCR exhibited genotypes A, a and a (Fig. 1). MBO REP 1 primer generated two banding patterns b and B (Fig. 1). Combining BOX A1R and MBO REP1 primers five genotypes were identified: ab (42%), aB (35%), Ab (15%), AB (7%) and ab (1%). Genotypes aB and ab were new and have not been reported in other studies using the same techniques. Whereas genotype aB was isolated from brood, extracted honey and honey supplies on brood combs, ab was detected only in extracted honey from one beekeeping operation with several bee yards near the Hungarian border. The occurrence of the different genotypes in the federal provinces of Austria is given in Table 1. Knowledge about the P. larvae genotypes in a country could be important for AFB risk assessment, because there is a correlation between genotype and occurrence of clinical symptoms. Genersch et al. (2005) demonstrated in exposure bioassays that genotype AB killed infected larvae faster than other genotypes. Because most larvae infected with genotype AB were dieing before cell capping, it could be hypothesized that nursing bees could detect and remove them, thus reducing the production of spores. Spreading of this genotype within the colony would therefore be slower and clinical AFB diagnosis would be handicapped because a spotty brood nest is dominating and fewer cells with perforated, sunken cappings, containing the ropy stage or foulbrood scales could be found. Consequently, unrecognized spreading of genotype AB in the field would be favored by beekeepers if they split colonies to increase hive numbers. In this study in two cases two different genotypes (ab and Ab) could be identified from the same comb of a colony. These combs came from two different beekeeping operations from two different federal provinces of Austria. A similar case was reported by Peters et al. (2006) for German P. larvae isolates, where two genotypes had been found in one apiary. Such multi-genotype infections of colonies or apiaries may increase the complexity of P. larvae infections by influencing type and speed of development of clinical symptoms.
Financial support was granted by the Federal Ministry of Agriculture, Forestry, Environment and Water Management according to Reg. (EC) No 797/2004 and the Austrian Agency for Health and Food Safety Ltd. (AGES). We thank ‘‘Biene Österreich” for support and Katharina Etter for technical assistance. References Alippi, A.M., Aguilar, O.M., 1998. Characterization of isolates of Paenibacillus larvae subsp. larvae from diverse geographical origin by the polymerase chain reaction and BOX primers. J. Invertebr. Pathol. 72, 21–27. Bakonyi, T., Derakhshifar, I., Grabensteiner, E., Nowotny, N., 2003. Development and evaluation of PCR assays for the detection of Paenibacillus larvae in honey samples: comparison with isolation and biochemical characterization. Appl. Environ. Microbiol. 69, 1504–1510. de Graaf, D.C., Alippi, A.M., Brown, M., Evans, J.D., Feldlaufer, M., Gregorc, A., Hornitzky, M., Pernal, S.F., Schuch, D.M., Titera, D., Tomkies, V., Ritter, W., 2006. Diagnosis of American foulbrood in honey bees: a synthesis and proposed analytical protocols. Lett. Appl. Microbiol. 43, 583–590. Forsgren, E., Stevanovic, J., Fries, I., 2007. Variability in germination and in temperature and storage resistance among Paenibacillus larvae genotypes. Vet. Microbiol. 129, 342–349. Genersch, E., Otten, C., 2003. The use of repetitive element PCR fingerprinting (rep PCR) for genetic subtyping of German field isolates of Paenibacillus larvae larvae. Apidologie 3 (2003), 195–206. Genersch, E., Ashiralieva, A., Fries, I., 2005. Strain- and genotype-specific differences in virulence of Paenibacillus larvae subsp. larvae, a bacterial pathogen causing American foulbrood disease in honey bees. Appl. Environ. Microbiol. 71, 7551– 7555. Genersch, E., Forsgren, E., Pentikäinen, J., Ashiralieva, A., Rauch, S., Kilwinski, J., Fries, I., 2006. Reclassification of Paenibacillus larvae subsp. Pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation. Int. J. Syst. Evol. Microbiol. 56, 501–511. Loncaric, I., Donat, C., Antlinger, B., Oberlerchner, J.T., Heissenberger, B., Moosbeckhofer, R., 2008. Strain-specific detection of two Aureobasidium pullulans strains, fungal biocontrol agents of fire blight by new, developed multiplex-PCR. J. Appl. Microbiol. 104, 1433–1441. Peters, M., Kilwinski, J., Beringhoff, A., Reckling, D., Genersch, E., 2006. American foulbrood of the honey bee: occurrence and distribution of different genotypes of Paenibacillus larvae in the administrative district of Arnsberg (North RhineWestphalia). J. Vet. Med. B Infect. Dis. Vet. Public Health 53, 100–104. Ritter, W., 1996. Diagnostik und Bekämpfung der Bienenkrankheiten. Gustav Fischer Verlag, Jena. Versalovic, J., Schneider, M., de Bruijn, F.J., Lupski, J.R., 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol. Cell. Biol. 5, 25–40.