Reclassification, Genotypes and Virulence of Paenibacillus Larvae

Reclassification, genotypes and virulence of Paenibacillus larvae, the etiological agent of American foulbrood in honeybees - a review Ainura Ashiralieva, Elke Genersch

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Ainura Ashiralieva, Elke Genersch. Reclassification, genotypes and virulence of Paenibacillus larvae, the etiological agent of American foulbrood in honeybees - a review. Apidologie, Springer Verlag, 2006, 37 (4), pp.411-420. hal-00892209

HAL Id: hal-00892209 https://hal.archives-ouvertes.fr/hal-00892209 Submitted on 1 Jan 2006

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie 37 (2006) 411–420 411 c INRA/DIB-AGIB/ EDP Sciences, 2006 DOI: 10.1051/apido:2006028 Review article

Reclassiﬁcation, genotypes and virulence of Paenibacillus larvae, the etiological agent of American foulbrood in honeybees – a review*

Ainura A,ElkeG

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

Received 20 December 2005 – revised 16 February 2005 – accepted 17 February 2005

Abstract – American foulbrood, a worldwide bacterial disease of honeybee brood caused by the gram- positive bacterium Paenibacillus larvae, is one of the most serious bee diseases. This review will focus on recent achievements in the study of Paenibacillus larvae brought about by molecular methods introduced into the field over the last fifteen years. One topic will be the classification of the etiological agent, which has changed several times since the first description in 1906 and was most recently modified again. Different genetic and biochemical methods for subtyping Paenibacillus larvae, the analysis of differences in virulence and the implications of these differences will also be covered.

American foulbrood / Paenibacillus larvae / taxonomy / genotyping / virulence

Abbreviations: AFB (American Foulbrood), P. l. larvae (Paenibacillus larvae subsp. larvae), P. l. pulvifaciens (Paenibacillus larvae subsp. pulvifaciens), P. l arvae (Paenibacillus larvae), CFU (colony forming units), LC50 (lethal concentration killing 50% of the larvae).

1. INTRODUCTION known as European foulbrood (EFB) caused by Melissococcus plutonius with B. alvei as The oldest report on foulbrood disease of a frequent secondary invader (Bailey, 1957; honeybees presumably dates back to Aristo- Bailey, 1983); on the other hand what was then tle (384–322 b.c.) who described in book IX called American foulbrood (AFB) with Bacil- of his History of Animals a diseased condi- lus larvae isolated as etiological agent (White, tion which “is indicated in a lassitude on the 1906). part of the bees and in malodorousness of the hive”. In 1769, the Saxon naturalist Schirach AFB is a serious bacterial disease of honey- described a honeybee disease characterized by bee brood, not only able to kill infected indi- a foul smell and coined the name foulbrood viduals but also potentially lethal to infected (Schirach, 1769). In 1885, the cause of foul- colonies. Spreading of the disease within an brood disease was ascribed to Bacillus alvei apiary and between apiaries or even countries (B. alvei) (Cheshire and Cheyne, 1885). In is facilitated by beekeeping practice like ex- 1906, it became evident that there were actu- changing material between colonies, manag- ally two different bacterial brood diseases to ing numerous hives in a confined area and the which the name foulbrood was being applied global trading of bees and honey. Meanwhile, (White, 1906): on the one hand a disease now AFB has spread worldwide. In many countries, AFB is a notifiable disease and most au- Corresponding author: E. Genersch, thorities consider burning of diseased colonies [email protected] and contaminated hive material the only work- * Manuscript editor: Gudrun Koeniger able control measure. Thus, AFB causes Article published by EDP Sciences and available at http://www.edpsciences.org/apido or http://dx.doi.org/10.1051/apido:2006028 412 A. Ashiralieva, E. Genersch considerable economic loss to beekeepers all agent exist in the literature (Katznelson, 1950; over the world. In some countries, antibiotics Gilliam and Dunham, 1978). There are also (e.g. oxytetracycline) are used to suppress the conflicting reports on the pathogenicity or vir- clinical symptoms and, hence, the outbreak of ulence of Bacillus pulvifaciens. Shortly after AFB. The drawbacks of this treatment are that Bacillus pulvifaciens had been isolated as the (i) antibiotics are not effective against spores causative agent of a honeybee larval disease, and, therefore, only mask the disease, and that its pathogenicity was questioned (Katznelson (ii) oxytetracycline-resistant P. la rv a e -strains and Jamieson, 1951) but then again confirmed have developed making the quest for new ef- by Hitchcock et al. (1979). Hence, Bacil- fective antibiotics already necessary (Miyagi lus pulvifaciens was considered a honeybee et al., 2000; Mussen, 2000; Kochansky et al., pathogen rarely causing any visible damage to 2001; Elzen et al., 2002; Wu et al., 2005). honeybees. Since the isolation and identification of When molecular methods were introduced the etiological agent of AFB in 1906 (White, into bacterial taxonomy it became evident that 1906), the disease has become one of the best- the genus Bacillus is phylogenetically very studied honeybee diseases (for a recent review heterogeneous. Comprehensive 16S rRNA see: Hansen and Brødsgaard, 1999). However, gene sequence analyses revealed that it con- many aspects of AFB remain elusive. In this sisted of at least five phyletic lines (Ash et al., review we will cover the great advances which 1991). One of these lines, the rRNA group 3 have been made over the past fifteen years by bacilli, was shown to be phenotypically and applying molecular methods to the study of phylogenetically sufficiently distinct to be as- AFB. We will especially focus on the new tax- signed to a separate genus, Paenibacillus (Ash onomic classification, on genotypes and differ- et al., 1993). Using a PCR probe test both, ences in virulence. Bacillus larvae and Bacillus pulvifaciens were identified as members of the new genus Paeni- bacillus and, hence, renamed Paenibacillus 2. RECLASSIFICATION larvae and Paenibacillus pulvifaciens, respec- OF THE ETIOLOGICAL AGENT tively (Ash et al., 1993). In 1996, the taxonomic position of Paeni- The bacterium causing AFB was first iden- bacillus larvae and Paenibacillus pulvifaciens tified and described by White (1906). He was again reviewed using a polyphasic ap- classified the bacterium that was consistently proach. Analysis of several type and refer- found in diseased and dead larvae as Bacillus ence strains of both species supported their re- larvae based on the rod-shaped morphology of classification into one species, Paenibacillus the vegetative form and the ability to sporulate larvae (Heyndrickx et al., 1996). Especially under adverse conditions. the rDNA restriction patterns and DNA-DNA In 1950, another bacterial disease of hon- binding studies revealed high levels of sim- eybee brood was described which was char- ilarity that did not support the former clas- acterized by larval remains forming pow- sification into two different species. How- dery scales rather than the hard scales ob- ever, at the infraspecific level phenotypic and served with AFB. Hence, the disease was genotypic differentiation into two subspecies, called powdery scale disease. The bacterium Paenibacillus larvae larvae (P. l. larvae)and isolated from those powdery scales resem- Paenibacillus larvae pulvifaciens (P. l. pulvi- bled Bacillus laterosporus and even more so faciens), seemed justified considering the dif- Bacillus larvae. On the basis of some char- ferent pathologies of the two former species acteristic differences it was nevertheless con- (Heyndrickx et al., 1996). The emended de- sidered a different species and named Bacil- scriptions of the two subspecies included some lus pulvifaciens (Katznelson, 1950). Powdery features differing between the subspecies. Be- scale disease turned out to be an extremely sides differences in pathogenicity, P. l. pulvi- rare condition. Only two reports describing faciens was described to differ from P. l. lar- the disease and the isolation of the etiological vae e.g. by a striking orange-pigmented colony P. l arvae and American foulbrood – a review 413

Table I. Former subspecies of P. l arvae and ERIC genotypes.

Paenibacillus larvae Genotypes acc. to ERIC-PCR ERIC I ERIC II ERIC III ERIC IV Colony morphology greyish orange-pigmented orange-pigmented greyish Former subspecies designation P. l. larvae problematic P. l. pulvifaciens P. l. pulvifaciens Diseased larvae show yes yes yes yes AFB-symptoms Pest-like disease progression yes yes questionable questionable Representatives referred to ATCC 9545T 03-194 ger LMG 16252 DSM 3615T in this manuscripta

Note: a Origin of strains: ATCC 9545T (American Type Culture Collection), 03-194 ger (German ﬁeld strain isolated from an AFB-outbreak in 2003), LMG 16252 (Belgian Type Culture Collection), DSM 3615T (German Type Culture Collection).

morphology not seen with P. l. larvae and by subspecies P. l. larvae and P. l. pulvifa- production of acid from mannitol but not from ciens as one species P. la rv a e without sub- salicin (Heyndrickx et al., 1996). However, al- species differentiation (Genersch et al., 2006). though the ability to produce an orange pig- The collection of field strains used for this ment was solely ascribed to P. l. pulvifaciens taxonomic study comprised not only non- (Heyndrickx et al., 1996), orange-pigmented pigmented but also orange-pigmented colony colonies isolated from diseased brood with variants of the former subspecies P. l. lar- symptoms of AFB had been reported ear- vae. All field and reference strains of the for- lier (Drobnikova et al., 1994) and recently it mer subspecies P. l. larvae had been iden- was demonstrated that this morphology char- tified using a subspecies specific PCR pro- acterizes a certain genotype of P. l. larvae tocol (Alippi et al., 2004) actually based on (Neuendorf et al., 2004). Hence, the validity genotyping via rep-PCR performed with ERIC of orange colony pigmentation as a distinc- primers (Versalovic et al., 1994). Typing of all tive feature was rebutted. Differentiation be- P. la rv a e strains with ERIC-PCR revealed that tween the two subspecies on the basis of their each former subspecies comprised two differ- differential acidification of salicin and man- ent genotypes resulting in a total of four P. la r- nitol (Heyndrickx et al., 1996) has also been vae ERIC-genotypes (Tab. I). The pigmented questionable since the fermentation of manni- strains of the former subspecies P. l. larvae tol and salicin has been reported to be a rather formed one of the four genotypes (Genersch variable property of field strains of P. l. lar- et al., 2006). Pulsed field-gel electrophoresis vae (Carpana et al., 1995; Dobbelaere et al., (PFGE) and SDS-PAGE profiling did not sup- 2001). The differentiation and classification of port the identity of the presumed P. l. pulvifa- the two subspecies became even more dubious ciens reference strains as a separate subspecies when the 16S rRNA gene sequences of DSM (Genersch et al., 2006). In contrast, the results 3615T, the type strain for P. l. pulvifaciens,and strongly suggested that the presumed P. l. pul- DSM 7030T, the type strain of P. l. larvae, vifaciens reference strains should be reclassi- were proven to be identical (Kilwinski et al., fied as proposed in Kilwinski et al. (2004). The 2004). strongest argument for the two different sub- Consequently, a most recent revision of the species had always been the different pathol- classification within the species Paenibacil- ogy of P. l. pulvifaciens (Katznelson, 1950; lus larvae using a polyphasic approach, in- Gilliam and Dunham, 1978). Therefore, ex- cluding not only type and reference strains posure bioassays were included in the revi- from different culture collections but also sion of the taxonomy of P. la rv a e (Genersch field strains from Germany, Finland, and Swe- et al., 2006). Honeybee larvae experimen- den, resulted in the reclassification of the tally infected with either P. l. larvae or P. l. 414 A. Ashiralieva, E. Genersch pulvifaciens showed disease symptoms and died, clearly proving that both former subspecies were pathogenic to honeybee larvae. Dead larvae developed into a ropy mass with a glue-like consistency and dried down to a hard scale irrespective of the presumed subspecies used for infection. Therefore, the described differences in pathology could not be verified in exposure bioassays (Tab. I). How- ever, clear differences in virulence could be observed for the analyzed P. la rv a e strains (Fig. 1). Those genotypes which belong to Figure 1. Time courses of infection for ERIC geno- the former subspecies P. l. pulvifaciens killed types (modified from Genersch et al., 2006). Hon- infected larvae very quickly as already sup- eybee larvae were experimentally infected with one posed earlier (Hitchock et al., 1979). It took representative of each of the four ERIC genotypes the former subspecies P. l. pulvifaciens only of P. l arvae using a spore concentration closest to around 2 days post infection to kill 50% and the estimated LC50. Mortality was monitored every 7 days post infection to kill 100% of the in- day. Cumulative mortality was calculated per day fected larvae (Genersch et al., 2006). There- post infection (p.i.) ± standard deviation (n = 3for fore, nearly all infected larvae died during their each strain) and expressed as percentage of infected larval stage before pupation and had been re- hosts (i.e. all larvae that died from AFB in this ex- moved by the nurse bees under natural con- periment). ditions. In contrast, ATCC 9545T, the type strain of the former subspecies P. l. larvae, the PCR-protocol described by Alippi et al. needed around 5 and 12 days post infection to kill 50% or 100% of the infected larvae, (2004) obviously allows for the identification of the P. la rv a e genotypes ERIC I and ERIC II respectively (Genersch et al., 2005; Genersch (Genersch et al., 2006) associated with clas- et al., 2006). In this case, nearly half of the sical AFB outbreaks. In combination with infected larvae died after pupation and had analysis of 16S rRNA (Govan et al., 1999) the chance to develop into a ropy mass and a or metalloprotease-genes (Neuendorf et al., hard scale under natural conditions. It is con- 2004), the PCR-based detection of P. la rv a e ceivable that these differences have an influ- provides a highly sensitive and specific and, ence on the probability of a pest-like disease therefore, extremely useful tool for the correct progression since the accumulation of spores laboratory diagnosis of AFB. and the spreading of the disease within a hive and between hives is dependent on the num- ber of spores produced. The more larvae die 3. SUBTYPING OF PAENIBACILLUS after pupation the more spores are produced LARVAE and the faster the disease will spread within the colony and between colonies. Following Epidemiological studies investigate the this line of thinking we would not expect rep- time and spatial distribution of infectious dis- resentatives of the former subspecies P. l. pul- eases. In most cases, the organisms causing vifaciens to cause classical AFB-outbreaks, al- an outbreak are clonally related and share though the finding of collapsed colonies with biochemical traits and genomic characteris- powdery scales (Katznelson, 1950; Gilliam tics. Bacterial subtyping, therefore, is use- and Dunham, 1978) may indicate that un- ful in determining the source of the infec- der certain conditions these P. la rv a e geno- tion, recognizing particularly virulent strains, types are also lethal to honeybee colonies and monitoring programs to combat AFB. The (Tab. I). Therefore, for diagnostic purposes it shortcomings of phenotypically based typ- ff may still be useful to di erentiate between ing methods have led to the development of ff the di erent genotypes of P. la rv a e . Applying molecular typing methods. P. l arvae and American foulbrood – a review 415

Table II. Subtyping of P. l arvae ERIC genotypes I and II with BOX A1R and MBO REP1 primers.

P. l a r v a e genotypes according to rep-PCR using ERIC- or BOX A1R/MBO REP1 primers ERIC-genotypesa ERIC I ERIC II BOX A1R/MBO REP1-genotypesb ab Ab aβ AB

Note: a Genersch et al., 2006; b Genersch & Otten, 2003.

First attempts to establish a molecular epi- total of four ERIC patterns within the species demiology of American foulbrood disease of P. la rv a e (Genersch et al., 2006). ERIC pat- honeybees were made a decade ago using tern II is congruent with the BOX/MBO geno- DNA restriction endonuclease fragments pat- type AB, while the other BOX/MBO geno- terns (REFP) (Djordjevic et al., 1994). Diges- types of the former subspecies P. l. larvae were tion of bacterial genomic DNA with Cfo Igen- shown to be subgroups of ERIC I (Tab. II). The erated useful REFPs and allowed the differen- type strains of the former subspecies P. l. lar- tiation of five clonal types of B. larvae (later vae (e.g. ATCC 9545T, DSM 7030T) belong P. l. larvae,nowP. la rv a e ) among geographi- to ERIC group I and to the fifth BOX/MBO cally diverse isolates. genotype aβ (Neuendorf et al., 2004). Repetitive element PCR fingerprinting, an- Recently, pulsed field-gel electrophoresis other method for molecular subtyping of bac- has also been used to genotype a range of terial isolates, also demonstrated considerable P. la rv a e isolates originating from Australia diversity within the former subspecies P. l. lar- and Argentina. Twelve distinct PFGE types vae (now P. la rv a e ). Using BOX primers three were identified among a total of 44 isolates closely related patterns were identified (Alippi (Wu et al., 2005). and Aguilar, 1998a) and confirmed with addi- Biochemical characterization of P. la rv a e tionally introduced primers REP (Alippi and using traditional macro (Jelinski, 1985; Alippi Aguilar, 1998b). ERIC primers were reported and Aguilar, 1998a) or commercial micro to produce no band differences within the for- methods (Carpana et al., 1995; Dobbelaere mer subspecies P. l. larvae or between former et al., 2001; Neuendorf et al., 2004) were also subspecies, P. l. larvae and P. l. pulvifaciens used in subtyping P. la rv a e . But it was not un- (Alippi and Aguilar, 1998b). However, later til genotyping was combined with a metabolic reports did demonstrate differences between fingerprinting technique (BIOLOG system) the two former subspecies, which even led that genotype-specific metabolic differences to the development of a “subspecies”-specific for P. la rv a e were demonstrated (Neuendorf PCR protocol (Alippi et al., 2004). et al., 2004). In this study, P. la rv a e geno- The analysis of a collection of German field type AB (ERIC II) exhibited the most strik- isolates also using rep-PCR but combining ing metabolic pattern, since it was the only primers BOX A1R and MBO REP1 (Genersch strain able to metabolize the carbohydrates D- and Otten, 2003) revealed the existence of at fructose and D-psicose, and the only strain un- least four genotypes (AB, Ab, ab, αB) caus- able to use glycerol as carbon source. In ad- ing AFB outbreaks in Germany. These geno- dition, genotype AB (ERIC II) was the only types showed remarkable constant geographic one harboring plasmid DNA (Neuendorf et al., clustering over the four year observation pe- 2004). These results showed for the first time ff ff riod. In this study, rep-PCR performed with that di erent genotypes of P. la rv a e also di er ERIC primers resulted in two banding pat- in phenotypic characteristics. terns, in contrast to earlier reports (Alippi and Aguilar, 1998b). It is now known that the for- 4. VIRULENCE OF PAENIBACILLUS mer subspecies P. l. larvae indeed splits up LARVAE into two ERIC patterns (ERIC I, II) and the former subspecies P. l. pulvifaciens splits up Varying definitions and uses of the terms in another two (ERIC III, IV) resulting in a pathogenicity and virulence exist in the 416 A. Ashiralieva, E. Genersch literature, especially in the discipline of in- vertebrate pathology (Thomas and Elkinton, 2004). A widely accepted definition indicates pathogenicity as a qualitative term describing the state of being pathogenic and virulence as a measure (i.e., the degree) of pathogenicity describing the disease producing power of a pathogen. For a given host and pathogen, pathogenicity is absolute whereas virulence is variable, e.g., due to strain or environmen- tal effects (Steinhaus and Martignoni, 1970; Shapiro-Ilan et al., 2005). Differences in virulence of a pathogen are best analyzed in ex- Figure 2. Time courses of infection for P. l arvae posure bioassays because it requires all steps BOX/MBO genotypes AB, Ab, ab,andaβ (modified of pathogenesis: entering the host, establishing from Genersch et al., 2005). Honeybee larvae were and reproducing, and causing disease. Three experimentally infected with several representatives common measures for virulence can be de- of each of the four BOX A1R/MBO REP1 subtypes β rived from such bioassays: LD50 or LC50,the of genotypes ERIC I (Ab, ab,anda ) and ERIC II respective dose or concentration it takes the (AB)ofP. l arvae (n = 6, 5, 5, 3 for AB, Ab, ab,and pathogen to kill 50% of the hosts tested, and aβ, respectively) using spore concentrations around LT 50, the estimated time it takes to kill 50% of the estimated LC50. Mortality was monitored every the infected individuals. day. The mean cumulative mortality per day post infection (p.i.) ± standard deviation was calculated It has been long known that the course of for each genotype. AFB in infected colonies can be quite variable. Although P. la rv a e is able to kill af- fected colonies, some infected colonies not only survive, but never develop clinical disease Some highly virulent strains killed 50% of the −1 symptoms visible to the apiculturist (Hansen larvae with less than 100 CFU mL larval and Brødsgaard, 1999). Hitherto, studies ad- diet, whereas it took the least virulent strain −1 dressing these differences in the outcome of tested around 800 CFU mL larval diet to AFB only focused on aspects of host tolerance kill 50% of the exposed larvae. Taking into ac- and hygienic behavior of honeybees (Tarr, count an additional factor of 2 coming in from ff 1938; Woodrow, 1942; Woodrow and Holst, the di erences in disease tolerance of the bees 1942; Sturtevant and Revell, 1953; Brødsgaard (Hoage and Rothenbuhler, 1966) spore con- et al., 1998; Hansen and Brødsgaard, 1999; centrations needed to cause clinical symptoms Brødsgaard et al., 2000; Spivak and Reuter, may vary with a factor of at least 20. These re- 2001). Indeed, a study directly comparing a sults indicate that from quantifying the spore susceptible with a resistant bee line revealed concentration present in brood comb honey it ffi that differences between bee strains might ac- is di cult to predict whether or not clinical count for a factor of 2 in the spore dose needed symptoms (ropy mass and foulbrood scales) for causing clinical symptoms in a colony will already be apparent in an infected colony. (Hoage and Rothenbuhler, 1966). However, a In the same study another reason was pre- most recent study using exposure bioassays sented to explain why spore concentration and for monitoring experimentally infected first in- visible clinical symptoms were not necessarily star bee larvae demonstrated that the impact correlated. Analysis of the time course of dis- of strain-specific differences in virulence of ease progression (Fig. 2) revealed genotype- P. la rv a e on spore dose is much greater than specific differences in the proportion of lar- the reported influence from bee tolerance to in- vae dying after cell capping (i.e. dying later fection (Genersch et al., 2005). The LC50sof than approximately six days post infection). different P. la rv a e strains, determined by ex- Since larvae which are moribund or dead be- posure bioassays, varied with a factor of 10. fore cell capping will most likely be removed P. l arvae and American foulbrood – a review 417

cate that irrespective of the spore concentration the likelihood to detect clinical symptoms is very low for infections caused by genotype AB (ERIC II) because most of the diseased larvae will be removed due to the hygienic behavior of the nurse bees. Nevertheless, the remaining larvae dying from the disease after cell capping are still sufficient to cause colony collapse and a pest-like disease progression as can be deduced from the fact, that this genotype is frequently isolated from AFB outbreaks in Germany, Sweden, and Finland (Genersch and Otten, 2003; Genersch et al., Figure 3. Correlation between time of larval death 2006; Peters et al., 2006). In contrast, infec- and genotype. Honeybee larvae were experimen- tions caused by any other BOX/MBO geno- tally infected with several representatives of each of type (ERIC I) can be diagnosed on the basis the four BOX A1R/MBO REP1 subtypes of geno- β of clinical symptoms at an early stage and, types ERIC I (a sgb,andAb) and ERIC II (AB)of hence, official measures can be taken directly. P. l arvae using spore concentrations covering the − − Because of these genotype-specific differences range between 100 CFU mL 1 and 2000 CFU mL 1 for each strain. Mortality was monitored every day. in virulence we recommend that genotyping of The mean value ± standard deviation of AFB-dead P. la rv a e isolated from colonies not showing larvae which died after cell capping was calculated clinical symptoms should be considered, espe- for each genotype. cially for countries allowing artificial swarm- ing as a possible control means (Fries et al., 2006), to enable the veterinarian to decide on by nurse bees, the typical clinical signs of AFB a rational basis for the most appropriate mea- like ropy mass in capped cells and foulbrood sures. scales only develop when larvae or pupae die after cell capping and are not cleaned out by nurse bees. Therefore, the more infected larvae 5. PERSPECTIVES die after cell capping, the more frequent clinical symptoms will be in an infected colony. Although the molecular pathogenesis of On the other hand, the more larvae die before American foulbrood is still far from being un- cell capping the less frequent clinical symp- derstood the work of the past fifteen years toms will be and the more difficult it will be to added a lot to the understanding of this dele- clinically diagnose AFB in such a case. Hence, terious bacterial disease of honeybee brood. it is remarkable that only 5.4 ± 3.2% of rep- With the establishment of P. la rv a e geno- resentatives of P. la rv a e BOX/MBO genotype types and their phenotypic differences includ- AB (ERIC II) were found to survive until af- ing variation in virulence, we are now in a sit- ter cell capping, whereas 26.6 ± 7.3, 20.2 ± uation where we can start to unravel certain as- 6.3, and 26.3 ± 2.8% of larvae infected with pects of the interaction between the pathogen BOX/MBO genotypes Ab, ab,andaβ, respec- P. la rv a e and its host, the honeybee larvae, at tively, died after capping (Fig. 3) (Genersch the molecular level. et al., 2005). The differences between the three genotypes Ab, ab,andaβ (all belong to ERIC I) and the genotype AB (ERIC II) when in- ACKNOWLEDGEMENTS fected larvae died were highly significant, with P values of 0.0006, 0.002, and 0.0004, respec- My thanks go to Kaspar Bienefeld who provided tively (one-way analysis of variance: df = 3, me with the admission ticket for bee pathology, to F = 14.06, P = 0.0002, followed by a post hoc Gudrun Koeniger who encouraged me to summa- test, Newman-Keuls test). These results indi- rize our recent findings, published in specialized 418 A. Ashiralieva, E. Genersch

journals, for the readers of Apidologie, and to col- Paenibacillus larvae / loque américaine / taxono- leagues past and present for fruitful cooperation and mie / virulence / caractérisation du génotype stimulating discussions.

Zusammenfassung – Paenibacillus larvae und die Amerikanische Faulbrut der Bienen – Reklas- sifizierung, Genotypen und Virulenz. Der Erre- Résumé – Reclassification, génotypes et viru- ger der Amerikanischen Faulbrut (AFB) wurde im lence de Paenibacillus larvae, agent de la loque Verlauf der Zeit bisher als Bacillus larvae, Pae- américaine des abeilles. L’agent de la loque amé- nibacillus larvae und Paenibacillus larvae larvae ricaine (AFB) a été classé au cours des temps klassifiziert. Parallel dazu wurde ein Bakterium, comme Bacillus larvae, Paenibacillus larvae et welches Larven tötet und zu Puderschorfen ein- Paenibacillus larvae larvae. En même temps une trocknen lässt, als Bacillus pulvifaciens, Paeniba- bactérie qui tuait les larves et les desséchait en cillus pulvifaciens und Paenibacillus larvae pulvi- croûtes poudreuses était caractérisée comme Bacil- faciens bezeichnet. Eine neuerliche molekulare Un- lus pulvifaciens, Paenibacillus pulvifaciens et Pae- tersuchung etlicher Typ- und Referenzstämme von nibacillus larvae pulvifasciens. L’étude moléculaire beiden Subspezies als auch von Feldisolaten er- des souches types et des souches de références des gab nun, dass es nur eine Spezies, Paenibacillus deux sous-espèces et des souches de terrain a mon- larvae, gibt, die mittels rep-PCR unter Verwen- tré qu’il s’agissait d’une seule espèce, Paenibacil- dung von ERIC-Primern in vier Genotypen diffe- lus larvae. L’analyse par PCR-répétitive à l’aide renziert werden kann. Experimentelle Infektions- dequatreamorcesERICapermisdedifférencier versuche zeigten keine unterschiedliche Patholo- quatre génotypes. Les tests d’infection expérimen- gie für die beiden Subspezies. Alle untersuchten tale n’ont montré aucune différence de pathologie Stämme waren pathogen für die Larven und zer- entre les deux sous-espèces. Toutes les souches étu- setzten die Larven zu fadenziehender Masse, die diées étaient pathogènes pour les larves et les dé- wiederum zu einem harten Schorf eintrocknete. In composaient en une masse filante qui se desséchait Deutschland kommen nur zwei der vier P. l ar- en une croûte dure. En Allemagne seuls deux des vae-Genotypen, wie sie mit ERIC-PCR dargestellt quatre génotypes de P. l arvae existent, comme le werden können, vor: ERIC I und ERIC II. Die- montre l’analyse par PCR-rep : il s’agit des gé- se Genotypen lassen sich noch weiter differenzie- notypes ERIC I et ERIC II. On les différencie en ren, wenn statt der ERIC-Primer BOX A1R- und combinant les amorces BOX A1R et MBO REP1. MBO REP1-Primer verwendet werden und diese Cette méthode permet de scinder encore plus le Ergebnisse kombiniert werden. Mit dieser Methode groupe ERIC I en deux. En Allemagne on trouve lässt sich die ERIC I-Gruppe weiter aufspalten, wo- principalement les génotypes BOX/MBO Ab, ab bei in Deutschland hauptsächlich die BOX/MBO- et αβ. Le groupe ERIC II est identique au gé- Genotypen Ab, ab und aβ vorkommen. ERIC II ist notype BOX/MBO AB.Lesdifférences entre les mit dem BOX/MBO-Genotyp AB identisch. Phäno- deux groupes sont principalement phénotypiques. typische Unterschiede bestehen hauptsächlich zwi- Le génotype AB/ERIC II présente une pigmenta- schen den beiden ERIC-Gruppen. So zeigen Kolo- tion orange sur gélose au sang de mouton et il est nien von AB/ERIC II eine orange Pigmentierung auf le seul à pouvoir métaboliser le fructose et