Reclassification, genotypes and virulence of Paenibacillus larvae, the etiological agent of American foulbrood in honeybees - a review Ainura Ashiralieva, Elke Genersch To cite this version: 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. pulvi- faciens (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 coun- tries, 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