Identification of Paenibacillus larvae to the subspecies level: An obstacle for AFB diagnosis

Journal of Invertebrate Pathology 91 (2006) 115–123 www.elsevier.com/locate/yjipa

IdentiWcation of Paenibacillus larvae to the subspecies level: An obstacle for AFB diagnosis Dirk C. de Graaf a,b,¤, Paul De Vos c, Marc Heyndrickx d, Stefanie Van Trappen e, Nico Peiren a, Frans J. Jacobs a a

Laboratory of Zoophysiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium b Veterinary and Agrochemical Research Centre, Brussels, Belgium c Laboratory of Microbiology, Ghent University, Ghent, Belgium d Department of Animal Product Quality and Transformation Technology, Agricultural Research Centre, Melle, Belgium e BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Ghent University, Ghent, Belgium Received 27 July 2005; accepted 24 October 2005 Available online 20 December 2005

Abstract This study was initially aimed at developing a PCR-test to diVerentiate between the pathogenic agent of American foulbrood (Paenibacillus larvae subsp. larvae) and powdery-scale disease (P. larvae subsp. pulvifaciens) of the honeybee. The test was based on the “insert of clone 9” (iC9), referring to a cloned 1.9 kB HaeIII fragment that occurs only in the P. larvae subsp. larvae reference strains and possibly correlates with American foulbrood virulence. It was shown that an iC9-based PCR-test discriminates between the BCCM/LMG reference strains of both subspecies. However, the screening of 179 Belgian Weld strains revealed Wve isolates that gave no iC9-based amplicon, thus rather resembling to P. larvae subsp. pulvifaciens. In addition, they all produced acid from mannitol, a characteristic previously assigned to the pulvifaciens subspecies. Because the reference strains gave conXicting data, this carbohydrate acidiWcation was not conclusive. Therefore, the exact taxonomic position of the Wve retained strains was determined by a polyphasic approach using SDS–PAGE, AFLP, and ERIC-based PCR. Four iC9-negative Weld strains could be identiWed as P. larvae subsp. larvae; the taxonomic position of the Wfth Weld strain remained ambiguous. The latter was provisionally classiWed as a subspecies pulvifaciens strain on the basis of SDS– PAGE. The present paper demonstrates the existence of Weld strains that do not Wt well in the subdivision of the species P. larvae into two subspecies. Knowing that only one of both subspecies represents the pathogenic agent of AFB, this is a serious obstacle for the diagnosis of this honeybee disease.  2005 Elsevier Inc. All rights reserved. Keywords: American foulbrood; Paenibacillus larvae; Honeybee; Apis mellifera; Diagnosis; Virulence determinant; Taxonomy

1. Introduction American foulbrood (AFB) is a disease of the brood of the honeybee, Apis mellifera, and is caused by the bacterium Paenibacillus larvae subsp. larvae (Pll). Within this species another subspecies P. larvae subsp. pulvifaciens (Plp) can be distinguished, which is associated with the socalled “powdery-scale disease” (Heyndrickx et al., 1996). Although both bee pathogens are uniWed at the species *

Corresponding author. Fax: +32 0 9 264 52 42. E-mail address: [email protected] (D.C. de Graaf).

0022-2011/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2005.10.010

level, they vary signiWcantly in pathogenic outcome, prevalence, and socio-economic impact. The powdery-scale disease is a very rare disease, with only a few documented cases and with no legal consequences. On the contrary, AFB aVects honeybee brood throughout the world, it is classiWed on list B of the OYce International des Epizooties (OIE) and is a notiWable, legal disease in most countries. In many countries, an eradication strategy exists with destruction of the infected beehives by burning when AFB has been diagnosed. According to the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Anonymous, 2004) the

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method to be applied for the diagnosis of AFB depends on the presence of clinical characteristics. In an advanced state of illness, AFB aVected brood combs are characterized by the glutinous consistency of the larval remains, which can be drawn out as threads with a thin device. If in addition a heat-treated sample of the putreWed larvae yields slowly growing colonies with a characteristic colony morphology, containing Gram positive, catalase negative rods, the conclusion will sound: AFB positive. When clinical signs are absent or information on the appearance of the brood is missing (for instance, the examination of honey or wax) the identiWcation of the pathogenic agent demands a more profound identiWcation of suspicious colonies. However, because of the very close relationship between the subspecies of P. larvae most DNA-linked molecular techniques do not allow a clear-cut discrimination between Pll and Plp. The present study was initially aimed at developing a PCR-test to diVerentiate between the AFB and powderyscale disease agents. It was based on RFLP analyses of Prince (2000) demonstrating that all BCCM/LMG culture collection strains of Pll showed a speciWc 1.9 kB HaeIII fragment that was not present in three tested strains of Plp. He cloned the fragment in a pGEM3Z(+) plasmide vector for sequencing (referred to as clone 9) and found the highest similarity between the “insert of clone 9” (iC9) and the Lactococcus lactis temperate bacteriophage, r1t. As it was found that typical virulent Pll strains carry two to three clone 9 hybridizing fragments, whereas a “hyper-virulent” strain contain two to three copies more, the 1.9 kB HaeIII fragment was supposed to correlate with AFB virulence. We will show that an iC9-based PCR-test can diVerentiate between the BCCM/LMG reference strains of Pll and Plp. However, the screening of 179 Belgian Weld strains revealed Wve isolates from which no iC9-based amplicon could be generated, thus rather resembling to Plp. As the phenotypic characterization was not conclusive, we performed SDS–PAGE, AFLP, and a PCR-test based on an enterobacterial repetitive intergenic consensus-PCR Wngerprint, in order to determine in a polyphasic approach their exact taxonomic position at subspecies level. In view of the discrepancy in control strategy of American foulbrood and powdery-scale disease, the occurrence of Pll strains that are diYcult to diVerentiate from Plp is a serious obstacle in the diagnosis of AFB.

and Stahly, 1983) at 37 °C and subcultures were administered to each contributing laboratory. Frozen stocks were deposited at the LMG research collection. The corresponding R-number will be used throughout the manuscript. Table 1 gives an overview of the studied strains, their aYliation and the techniques applied. All strains were grown aerobically on MYPGP plates (PCR, growth inhibition test, and low temperature growth) or Colombia agar supplemented with 5% sterile sheep blood (cell and colony morphology, biochemical proWling, and AFLP). Incubation was at 37 °C, except for the low temperature growth (20 °C) and the biochemical proWling of the Plp reference strains (28 °C, corresponding to their optimal growth condition). When recovered from the research collection for the performance of the microbiological tests, all Weld strains showed a homogeneous colony type, except for strain R 20833 that resulted in two colony types. Both types were tested separately. 2.2. Polymerase chain reaction (PCR) A PCR-test was developed for rapid screening of bacterial strains for the presence of the iC9 fragment. Oligonucleotide primers were designed using the Vector NTI software (Informax, Inc.) resulting in a 5⬘-CCAGCTC AAAATCCTACGGT-3⬘ forward primer and a 5⬘-CATG GGATTTTCTTCGGGAG-3⬘ reverse primer that covered the Wrst 606 bp of iC9. Template DNA was prepared by heating (95 °C) a bacterial suspension for 15 min (Dobbelaere et al., 2001a). The 50
l reaction mixtures contained 10.0
l Q-solution (Qiagen), 5.0
l of 10£ PCR buVer providing 1.5 mM MgCl2, 1.25 U HotStar Taq polymerase (Qiagen), 50 pmol of both forward and reverse primer, 10 nM of each dNTP (Gibco BRL) and 5.0
l of template DNA. Following an initial step at 95 °C for 15 min, the reaction mixtures were subjected to 30 cycles consisting of denaturation at 93 °C for 1 min, primer annealing at 55 °C for 30 s, and DNA extension of 1 min at 72 °C. The reactions were completed by a Wnal extension at 72 °C for 5 min. In addition, two known PCR-tests were done as previously described: (i) the test based on the 16S rRNA gene, proven to be speciWc till the species level (Dobbelaere et al., 2001a) and (ii) a recently developed test based on an enterobacterial repetitive intergenic consensus-PCR Wngerprinting method (ERIC) (Alippi et al., 2004).

2. Materials and methods 2.3. Phenotypic characterization 2.1. Bacterial strains and growth conditions All reference strains were from the BCCM/LMG culture collection. Field strains of P. larvae were isolated from Belgian honey samples during an epidemiological survey (de Graaf et al., 2001) or from the routine diagnosis of bee diseases at the Veterinary and Agrochemical Research Centre in Brussels, where they were preserved as frozen cells. They were recovered on MYPGP (the abbreviation refers to its constituents: Mueller–Hinton broth, yeast extract, potassium phosphate, glucose, and pyruvate) plates (Dingman

Cell and colony morphology of all strains were described and their biochemical proWle was determined using the API 50 CHB kit (BioMérieux). Inoculation and incubation of the latter were done according to manufacturer’s instructions. The tests were performed in duplo: ones following the standard protocol using a bacterial suspension at moderate density (McFarland 2) and ones using a more dense suspension (McFarland 5). The strips were incubated aerobically at 37 or 28 °C (see growth conditions) and were read after 24 and 48 h.

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117

Table 1 List of strains studied Species

Subsp.

Bacillus cereus Bacillus licheniformis Bacillus megaterium Bacillus subtilis Bacillus thuringiensis Brevibacillus laterosporus Enterococcus laterosporus Melissococcus plutonius Paenibacillus apiarius Paenibacillus larvae larvae

Strain no.

Other designation

Source

LMG 6923T LMG 6933T LMG 7127T LMG 7135T LMG 7138T LMG 16000T LMG 7937T LMG 17433T LMG 9820T

D ATCC 9545T D DSM 7030T

Foulbrood of honeybees

D NRRL B-2605T

Applied techniques iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP, SDS–PAGE

D LMG 15969T Paenibacillus larvae

larvae

LMG 14425

Paenibacillus larvae Paenibacillus larvae Paenibacillus larvae Paenibacillus larvae

larvae larvae larvae pulvifaciens

LMG 15969T LMG 16245 LMG 18149 LMG 6911T

D ATCC 25747

Ohio, USA, diseased honeybee larvae See LMG 9820T See LMG 9820T D NRRL B-3650 Diseased honeybee larvae D Hornitzky 89/2302/4 Victoria, Australia, honeybee D ATCC 13537T Dead larvae honeybee D DSM 3615T

iC9-PCR, 16S-PCR, ERIC-PCR, MT, BP, SDS–PAGE iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR SDS–PAGE SDS–PAGE

D IFO 15408T D NCIMB 11201T D NRRL B-3688T D NRRL B-3685T D NRRL B-3670T D LMG 16248T D LMG 15974T Paenibacillus larvae

pulvifaciens LMG 14427

D ATCC 25367

Paenibacillus larvae Paenibacillus larvae

pulvifaciens LMG 14428 D ATCC 25368 pulvifaciens LMG 15974T See LMG 6911T

Unknown See LMG 6911T

Paenibacillus larvae Paenibacillus larvae Paenibacillus larvae Paenibacillus larvae

pulvifaciens pulvifaciens pulvifaciens pulvifaciens

(1949), honeybee larvae See LMG 6911T Unknown Unknown

iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP, SDS–PAGE iC9-PCR, 16S-PCR, SDS–PAGE iC9-PCR, 16S-PCR, ERIC-PCR, MT, BP, SDS–PAGE iC9-PCR, 16S-PCR SDS–PAGE SDS–PAGE SDS–PAGE

D DSM 8443 D NRRL NRS-1684

Dead honeybee larvae

SDS–PAGE

R 20833 t1

Honey 2 spores/5 g

iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP, SDS–PAGE iC9-PCR, 16S-PCR, ERIC-PCR, MT, BP, SDS–PAGE iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP, SDS–PAGE iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP, SDS–PAGE iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP, SDS–PAGE iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP, SDS–PAGE iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP, SDS–PAGE iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP, SDS–PAGE

LMG 16247 D NRRL B-368 LMG 16248T See LMG 6911T LMG 16250 D NRRL B-14154 LMG 16251 D CCM 38 D CCUG 7427

Unknown

D NCFB 1121 D NRRL NRS-1283 Paenibacillus larvae Paenibacillus larvae

pulvifaciens LMG 16252 Ho 11

R 20833 t2 Paenibacillus larvae

Ho 31

R 20834

Honey >50 spores/5 g

Paenibacillus larvae

Ho 485

R 20828

Honey 4 spores/5 g

Paenibacillus larvae

Ho 908

R 20829

Honey 35 spores/5 g

Paenibacillus larvae

Ho 1326

R 20835

Honey 1 spore/5 g

Paenibacillus larvae

Ho 1338

R 20830

Honey 4 spores/5 g

Paenibacillus larvae

Ho 1348

R 20836

Honey >50 spores/5 g

Paenibacillus larvae

LIMS 6316/5 R 20837

Brood subclinical AFB

iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP, SDS–PAGE (continued on next page)

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Table 1 (continued) Species Paenibacillus larvae

Subsp.

Strain no. LIMS 6754

Other designation R 20831

Source Brood subclinical AFB

Paenibacillus larvae

LIMS 7522

R 20832

Brood clinical AFB

Paenibacillus lautus Paenibacillus polymyxa Paenibacillus validus

LMG 11157T LMG 13296T LMG 9817

Applied techniques iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP iC9-PCR, 16S-PCR, ERIC-PCR, MT, GIA, LT°, BP, AFLP, SDS–PAGE iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR iC9-PCR, 16S-PCR

Abbreviations (in order of appearance): LMG, BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie Gent, Universiteit Gent, Belgium; ATCC, American Type Culture Collection, Rockville, Maryland, USA; DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany; NRRL, Agricultural Research Service Collection, Illinois, USA; IFO, Institute for Fermentation, Osaka, Japan; NCIMB, The National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland, UK; CCM, Czechoslovak Collection of Microorganisms, Brno, Czech Republic; CCUG, Culture Collection University of Göteborg, Göteborg, Sweden; NCFB, National Collection of Food Bacteria, Aberdeen, Scotland, UK; R, Research Collection of LMG; Ho, honey sample from a Belgian epidemiological survey (de Graaf et al., 2001); LIMS, Laboratory Information Management System, Veterinary and Agrochemical Research Centre, Brussels, Belgium; t1, type 1; t2, type 2; MT, basic microbiological tests; GIA, growth inhibiting activity against LMG 9820T; LT°, low temperature growth; BP, biochemical proWling.

Growth inhibiting activity against Pll reference strain LMG 9820T was done by plating the test strain next to the reference strain on the same MYPGP plate, leaving only a sharp zone in between. Two days later the assay was evaluated. The test was considered positive when a zone of growth inhibition was observed of more than 5 mm on the side of the LMG 9820T strain. 2.4. AmpliWed fragment length polymorphism (AFLP) AFLP was performed with the restriction enzymes (EcoRI and MseI) and primers with selective nucleotides (C and A, respectively) as described previously (Heyndrickx et al., 1996), with some modiWcations described as follows. A non-selective ampliWcation preceded the selective ampliWcation using adapter-speciWc primers without selective nucleotides and a PCR-program consisting of a Wrst step at 72 °C for 2 min and 20 cycles of denaturation at 94 °C for 20 s, annealing at 56 °C for 45 s, and elongation at 72 °C for 2 min. In the selective ampliWcation, 1.5
l of Wve times diluted preselective PCR-product was used as template. Both non-selective and selective ampliWcations were performed in ampliWcation core mix (Applied Biosystems). Furthermore, radioactive labelling of the selective EcoRI-primer was replaced by Xuorescent 6-FAM labelling (Applied Biosystems) and separation and detection of the denatured fragments was achieved on a capillary sequencer (ABI PRISM 310) using the POP4 separating polymer on a 47 cm £ 50
m capillary (Applied Biosystems). AFLP Wngerprints were imported from Genescan version 3.1 (Applied Biosystems) into BioNumerics 3.0 (Applied Maths) and clustered using the Pearson correlation coeYcient and the unweighted pair group method (UPGMA) clustering algorithm.

interpretation as described by Vauterin and Vauterin (1992). 3. Results 3.1. iC9-based and 16S rRNA-based PCR The Pll strains LMG 9820T, LMG 14425, LMG 15969T, and LMG 16245 gave the expected amplicon of 606 bp in the iC9-based PCR-test, whereas no amplicon was obtained from the Plp strains LMG 14427, LMG 14428, LMG 15974T, and LMG 16247. All eight reference strains generated the expected 973 bp amplicon in the 16S rRNA-based PCR-test. Thereafter, the iC9-based PCR was tested for crossreactions with bacterial species closely related to P. larvae and/or described to occur in beehives: Bacillus cereus LMG 6923T, Bacillus licheniformis LMG 6933T, Bacillus megaterium LMG 7127T, Bacillus subtilis LMG 7135T, Bacillus thuringiensis LMG 7138T, Brevibacillus laterosporus LMG 16000T, Enterococcus laterosporus LMG 7937T, Mellisococcus plutonius (kindly provided by Dr. B. Ball, Rothemsted, UK), Paenibacillus apiarius LMG17433T, Paenibacillus lautus LMG 11157T, Paenibacillus polymyxa LMG 13296T, and Paenibacillus validus LMG 9817. None of them resulted in any amplicon under the speciWed PCR conditions. The iC9-based PCR-test was further evaluated by screening our complete Weld strain collection of 145 isolates from honey (de Graaf et al., 2001) and 34 isolates from clinical and subclinical Belgian cases of AFB, all identiWed as P. larvae based on a positive 16S rRNA-based PCR. Surprisingly, from Wve isolates the expected 606 bp iC9 PCRproduct could not be obtained: R 20833, R 20834, R 20835, R 20836, and R 20837.

2.5. SDS–PAGE of whole-cell proteins 3.2. Phenotypic characterization After an incubation period of 24 h, preparations of whole-cell proteins and SDS–PAGE were performed as described by Pot et al. (1994) and the data collection and

Basic microbiological tests were done with 14 retained P. larvae strains only: the Wve iC9-PCR negative Weld

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strains, Pll strains LMG 9820T and LMG 14425, Plp strains LMG 14427, and LMG 15974T, and Wve randomly chosen iC9-PCR positive Weld strains. The results are given in Table 2. All examined bacteria were rods with a width of about 1.0
m and a length between 1.5 and more than 20
m. They all appeared as single cells or pairs, sometimes as short chains. Two Weld strains were motile (R 20835 and R 20836) and likewise was the Plp reference strains (LMG 14427). All colonies (except R 20833t2) were beige coloured, variable in size (diameter varied between 0.8 and 2.0 mm), Xat or raised and mostly rough. The Plp reference strain LMG 14427 was positive in the growth inhibition test and grew at 20 °C, in contrast to the Weld strains. The same 14 strains were also tested for carbohydrate acidiWcation. The most invariant results were obtained when a dense bacterial suspension was loaded on API 50 CHB strips and read after 48 h. Only these data are given below (Table 3). All strains tested negative for erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, adonitol, -methyl-xyloside, L-sorbose, rhamnose, dulcitol, inositol, sorbitol, -methyl-D-mannoside, amygdaline, arbutine, esculine, cellobiose, lactose, melibiose, saccharose, inuline, melezitose, D-raYnose, amidon, glycogene, xylitol, -gentiobiose, D-turanose, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, and 2-keto-D-gluconate. All strains were positive for N-acetylglucosamine. The outcome of the other carbohydrates diVered considerably among the strains. Based on the proWle of the four reference strains, there were no characteristic reactions for one of the subspe-

119

cies, except -methyl-D-glucoside that gave the same negative result for the two Pll references, but here the two Plp references tested “ambiguous”. Further we noticed that all iC9-PCR negative Weld strains tested positive for mannitol, but the outcome of the reference strains was not equivocal. 3.3. AFLP In the clustering of AFLP Wngerprints (Fig. 1), no clear separation between the Wve iC9-PCR negative and positive strains was achieved. On the contrary, all strains included showed a strain-speciWc Wngerprint. The reference strains of Pll and Plp were well separated from each other on the basis of polymorphic bands which corresponded well with those in the original AFLP Wngerprints reported by Heyndrickx et al. (1996), but these reference strains were also clearly diVerent from the Weld strains included. Only two iC9-PCR negative Weld strains (R 20833 and R 20834) showed similar AFLP Wngerprints at a similarity of 83% with a third iC9-PCR negative strain (R 20837) linking at a similarity level of 76%. 3.4. SDS–PAGE of whole-cell proteins Protein proWles of the subset of 14 strains showed a division in two groups clustering at a similarity level of 78% (upper group) and 82% (lower group) (Fig. 2). The upper group contained all Plp reference strains and one iC9-PCR negative Weld strain (R 20835), the former being clustered at

Table 2 Basic microbiological tests of Paenibacillus larvae reference strains and Weld strains Strains studied

LMG 9820T

LMG 14425

LMG 15974T

LMG 14427

R 20828

R 20829

R 20830

R 20831

R 20832

R 20833 t1

R 20833 t2

R 20834

R 20835

R 20836

R 20837

Subspecies iC9-based PCR

Pll +

Pll +

Plp ¡

Plp ¡

+

+

+

+

+

¡

¡

¡

¡

¡

¡

Cell size Widtha Min. length Max. length

0.9 1.5 5.0

1.0 3.0 >20.0

1.0 3.0 >13.0

1.0 3.0 13.0

1.0 2.0 >20.0

1.0 3.0 >15.0

1.0 4.0 >20.0

1.0 4.0 >20.0

1.0 2.0 >20.0

1.0 2.0 20.0

1.0 2.0 20.0

1.0 2.0 >15.0

1.0 2.0 >20.0

1.0 2.0 >20.0

1.0 2.0 >20.0

Appearanceb Short chains Motile

y n

n n

n n

n y

y n

n n

y n

y n

y n

y n

y n

y n

y y

y y

y n

ND F S

0.8 F Ro

1.5 Ra Ro

1.5 Ra Ro

0.9 F

0.9–1.2 F

0.7–0.9 F

1.0–1.5 Ra Ro

2.0 Ra Ro

1.0 Ra

1.0 Ra

1.0 F

1.0 F

0.4 F

1.5 Ra Ro

B ¡ ¡

B ND ND

B ND ND

B + +

B ¡ ¡

B ¡ ¡

B ¡ ¡

B ¡ ¡

B ¡ ¡

T B ¡ ¡

W ND ND

B ¡ ¡

B ¡ ¡

B ¡ ¡

B ¡ ¡

Colony Diameterc Flat/raised Smooth/rough Translucent Colour Growth inhib.d Low°t growth

Abbreviations: LMG, BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie Gent, Universiteit Gent, Belgium; R, LMG Research Collection; Pll, Paenibacillus larvae subsp. larvae; Plp, Paenibacillus larvae subsp. pulvifaciens; ND, not done; F, Xat; Ra, raised; S, smooth; Ro, rough; T, translucent; B, beige; W, white. All vegetative cells were rods that appeared single or in pairs. No spores were formed after 1 day. All colonies were circular and undulate. a In
m. b y, yes; n, no. c In mm. d Growth inhibiting activity against LMG 9820T.

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Table 3 Biochemical proWle of Paenibacillus larvae reference strains and Weld strains Strains studied

LMG 9820T

LMG 14425

LMG LMG 15974T 14427

R R R R R R R R R R R 20828 20829 20830 20831 20832 20833 t1 20833 t2 20834 20835 20836 20837

Subspecies iC9-based PCR Glycerol Ribose Galactose D-Glucose D-Fructose D-Mannose Mannitol -Methyl-D-glucoside Salicine Maltose Trehalose D-Tagatose 5-Keto-D-gluconate

Pll + +a + ¡ + + + § ¡ + ¡ + ¡ ¡

Pll + § § ¡ § ¡ § ¡ ¡ § ¡ + § §

Plp ¡ + + ¡ + ¡ + + § § ¡ + § §

+ § + ¡ + ¡ § ¡ ¡ ¡ ¡ + § ¡

Plp ¡ § + § + § § § § § § + § ¡

+ § § ¡ + § § ¡ ¡ § ¡ + § §

+ § + ¡ + ¡ + ¡ ¡ ¡ ¡ + § §

+ + + ¡ + § § ¡ ¡ § ¡ + § §

+ + § ¡ + § § ¡ ¡ § ¡ § § §

¡ ¡ + ¡ + + + + ¡ § § + § §

¡ ¡ + ¡ + + + + ¡ § § + § ¡

¡ ¡ + ¡ + + + + ¡ § ¡ + § ¡

¡ ¡ + ¡ + + + + ¡ § ¡ + § ¡

¡ ¡ + ¡ + + + + ¡ + ¡ + § ¡

¡ ¡ § ¡ + + + + ¡ ¡ ¡ § § ¡

All strains were negative for erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, adonitol, -methyl-xyloside, L-sorbose, rhamnose, dulcitol, inositol, sorbitol, -methyl-D-mannoside, amygdaline, arbutine, esculine, cellobiose, lactose, melibiose, saccharose, inuline, melezitose, D-raYnose, amidon, glycogene, xylitol, -gentiobiose, D-turanose, D-lyxose D-fucose, L-fucose, D-arabitol, D-arabitol, gluconate, and 2-keto-D-gluconate. All strains were positive for N-acetylglucosamine. a +, positive; §, ambiguous; ¡, negative.

Fig. 1. Dendrogram based on UPGMA clustering of correlation coeYcients of normalized computer proWles from AFLP analyses of Paenibacillus larvae reference strains and Weld strains.

82% similarity. The lower group contained all Pll reference strains and the remaining Weld strains. In this lower group another subdivision could be recognized: one with the three remaining iC9-PCR negative Weld strains clustering with the Pll type strain (LMG 9820T) and one with all iC9-PCR positive Weld strains clustering with the other Pll reference strains (LMG 18149 and LMG 14425). 3.5. ERIC-based PCR-test The ERIC-based PCR-test gave a clear diVerentiation at the subspecies level of tested P. larvae reference strains: a PCR amplicon of the expected size of ca. 550 bp was only found in the larvae subspecies. The 10 Weld strains were all positive, although some of them gave only a weak band on agarose gel-electrophoresis (not shown). 4. Discussion The close taxonomic relationship of Pll and Plp is an important issue in the diagnosis of AFB. Despite the intro-

duction of advanced molecular techniques in bee pathology, only limited progress has been made on the diVerentiation of these two subspecies. Most PCR-tests are based on conserved target genes (16S rRNA) and initially these tests could only distinguish the species P. larvae from other bacterial species (Govan et al., 1999; Dobbelaere et al., 2001a; Lauro et al., 2003). Later on, 16S rRNA based PCRs were more or less Pll speciWc by addition of restriction fragment analysis (Alippi et al., 2002), reducing the number of cycles (Piccini et al., 2002) or selecting unique primer sets (Kilwinski et al., 2004). The use of molecular tools for the identiWcation of pathogenic agents that are designed on virulence factors or their genes, are very common in medical diagnosis (Thorne, 1992). They oVer the advantage not only to identify a microbial organism, but also to predict its pathogenic power. It was suggested that metalloproteases from Pll play a role in the virulence of the bacterium (Holst and Sturtevant, 1940; Jarosz and Glinski, 1990; Dancer and Chantawannakul, 1997) and these enzymes and their genes have been employed successfully for the diagnosis of AFB. Already in 1940 the clotting of

D.C. de Graaf et al. / Journal of Invertebrate Pathology 91 (2006) 115–123

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Fig. 2. Dendrogram based on UPGMA clustering of correlation coeYcients of normalized computer proWles from SDS–PAGE analyses of whole-cell proteins from Paenibacillus larvae reference strains and Weld strains.

milk by AFB bacteria due to proteolytic cleavage has been described (Holst and Sturtevant, 1940), and recently a PCR-test based on a 35 kDa metalloprotease was proven to be subspecies speciWc (Kilwinski et al., 2004). The iC9 fragment seemed another interesting target for an American foulbrood speciWc PCR-test. According to Prince (2000) (i) this 1.9 kB HaeIII fragment occurs in the subspecies larvae reference strains only, (ii) the copy number is possibly related to virulence, and (iii) sequence analysis in the vicinity of this region found homology to YopB, which is a virulence determinant of the type III secretory mechanism of Yersinia pestis (Cornelis and Wolfwatz, 1997). The PCR-test we developed can distinguish the BCCM/ LMG reference strains of Pll from those of Plp. However, with the Wnding of Wve P. larvae Weld strains that were iC9PCR negative, the exact taxonomic position of these bacteria till the subspecies level was required for Wnal evaluation of the test. It was unexpected that some of these Belgian Weld strains responded to the iC9-PCR in the same negative way as the references strains of Plp, thus implying they should be identiWed as such. Indeed, (i) powdery-scale disease has so far not been observed in Belgium and (ii) clinical signs of this disease were absent (brood comb-derived strains) or missing (honey-derived strains) for each of the Wve iC9-PCR negative Weld strains. Although recently a few rapid PCR-tests have been described from which it is claimed that they can diVerentiate between Pll and Plp (Alippi et al., 2004; Kilwinski et al., 2004), we return back to the techniques applied in the original taxonomic study of Heyndrickx et al. (1996) for assignment of the subspecies involved. Indeed, the uniWcation of P. larvae subsp. larvae (formerly Bacillus larvae) and P. larvae subsp. pulvifaciens (formerly Bacillus pulvifaciens) at the species level and their

separation at the subspecies level was the result of a polyphasic approach comprising of basic microbiological tests, low temperature growth, biochemical proWling, SDS– PAGE and AFLP (Heyndrickx et al., 1996). In the present study, exactly the same techniques were performed for typing the ambiguous Weld strains, completed with the growth inhibition assay and the ERIC-PCR. Phenotyping of the retained Weld strains could not conWrm whether they were really of the pulvifaciens subspecies or not. Moreover, the iC9 negative strains showed some characteristics previously assigned to the larvae subspecies: growth inhibition negative and no growth at low temperature. Prince (2000) demonstrated that Plp produces a heatlabile bacteriocin during growth-phase, which is speciWcally active and lytic against Pll and B. subtilis. This bacteriocin is diVerent from the heat-stabile polycyclic, macrolide antibiotic “apidiothricin” produced by Pll during sporulation (Neumann et al., 1997). Our inhibition test was designed for screening growth competition against a Pll reference (LMG 9820T) before sporulation occurs, thus pointing to a bacteriocin activity possibly coming from Plp. However, in other bacilli the production of bacteriocins or bacteriocin-like compounds was proven to be highly speciWc at strain level, more than at the species level (Martirani et al., 2002). Therefore, our results of the growth inhibition test should be considered with care. On the contrary, growth at low temperature has so far not been brought into question and was considered a typical characteristic of Plp from its Wrst description on (Katznelson, 1950). The diVerentiation of the subspecies on the basis of carbohydrate acidiWcation was much more diYcult and no clear diVerential pattern could be found. The characteristic biochemical proWle for the species P. larvae with acid from

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glucose and trehalose and no acid from arabinose and xylose (Gordon et al., 1973; Carpana et al., 1995) was fulWlled for all tested strains (only LMG 14425 gave “ambiguous” for D-glucose), but the diVerential proWle for Plp given by Heyndrickx et al. (1996) with acid from mannitol and no acid from salicine, could not be conWrmed. Our iC9-PCR negative strains were all positive for mannitol, but as the reference strains gave conXicting results these data should be considered with great care. In addition, several research groups found that the acidiWcations of mannitol (Dobbelaere et al., 2001b; Gordon et al., 1973) and salicine (Carpana et al., 1995) were rather variable properties of P. larvae. The molecular Wngerprinting techniques could only partially give us the taxonomic position of the Wve retained strains. AFLP failed to do so, and this was unexpected as it was applied with success in the study of Heyndrickx et al. (1996). May be the AFLP proWle of at least the Pll and possibly the Plp reference strain is not representative for the genetic diversity found in Weld strains. SDS–PAGE gave a clear-cut discrimination of all tested Plp and Pll reference strains, and based on their SDS–PAGE proWle (together with a positive ERIC-PCR and no growth at low temperature) four of the iC9-negative strains could deWnitely be identiWed as Pll: R 20833 (types 1 and 2), R 20834, R 20836, and R 20837. The taxonomic position of R 20835 remains ambiguous because on the one hand it clustered with the Plp reference strains in SDS–PAGE and on the other hand it was ERIC-PCR positive and failed to growth at low temperature. Provisionally it was classiWed as a subspecies pulvifaciens strain because of its SDS–PAGE proWle. Not only Wts the overall band pattern of R 20835 with that of Plp, but in addition this strain contains the unique dense band described by Heyndrickx et al. (1996) in the upper part of the proWles of Plp protein extracts. Recently, Kilwinski et al. (2004) suggested that the Plp type reference strain DSM 3615T ( D LMG 6911T D LMG 15974T D LMG 16248T) should be reclassiWed as Pll based on biochemical and molecular characteristics. In the present paper, the synonym strain LMG 6911T was found to cluster in SDS–PAGE with the other Plp reference strains, being a major argument against such a reclassiWcation. The present paper demonstrates the existence of Weld strains that do not Wt well in the subdivision of the species P. larvae into two subspecies. Knowing that only one of both subspecies represents the pathogenic agent of AFB, this is a serious obstacle for the diagnosis of this honeybee disease. In addition, in the past decade there seems to be a tendency to narrow the separation between the two subspecies. For instance, orange pigmented or weak, delayed catalase positive P. larvae strains have previously been ruled out as Pll and now it has been openly questioned whether at least some of them were misdiagnosed (Kilwinski et al., 2004; Neuendorf et al., 2004). In the present study the subspecies speciWcity of growth at low temperature and the ERIC-PCR was brought up for discussion. However, in a way it is inherent in large scale screening of Weld strains that some atypical strains might emerge.

Are the atypical strains that we found in this study of any clinical signiWcance? Today, we can only know by testing their pathogenicity on living larval material. However, on the long term the solution should come from the study of the virulence determinants of the etiological agent of American foulbrood. Information about the molecular mechanism of the attachment to and invasion across the midgut cell lining, the survival and passage through the epithelial cell cytoplasm, the invasion in the haemocoel and the escape of the immunity of the honeybee larvae, will provide us more precise tools to be employed in the diagnosis of AFB. EVorts are underway to generate whole-genome sequence data for P. larvae subsp. larvae (at the Baylor College of Medicine Human Genome Sequencing Center, USA) and smaller-scale eVorts could easily generate dozens of plausible virulence related genes. This could lead to a diVerentiated policy against AFB, that takes into account the virulence of the Pll strains involved. Concerning the iC9 fragment, we have conWrmed that it is abundantly distributed in P. larvae Weld strains. Because the developed iC9-PCR seems to be highly Pll speciWc (so far no false positive reactions were found) it could be helpful to unravel taxonomic questions. However, because of its rather low sensitivity (approximately 93%) the iC9-PCR can never be used alone, but should be done in combination with other tests. The present study provides no information about the involvement of the iC9 fragment in the pathogenicity of Pll. But this issue will receive our attention in the near future. Acknowledgment S.V.T. was supported by the Federal Public Planning Service—Science Policy, Belgium. References Alippi, A.M., Lopez, A.C., Aguilar, O.M., 2002. DiVerentiation of Paenibacillus larvae subsp. larvae, the cause of American foulbrood of honeybees, by using PCR and restriction fragment analysis of genes encoding 16S rRNA. Appl. Environ. Microbiol. 68, 3655–3660. Alippi, A.M., Lopez, A.C., Aguilar, O.M., 2004. A PCR-based method that permits speciWc detection of Paenibacillus larvae subsp. larvae, the cause of American foulbrood of honey bees, at the subspecies level. Lett. Appl. Microbiol. 39, 25–33. Anon., 2004. American foulbrood. In: OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, fourth ed. OYce International des Epizooties, Paris, pp. 970–978. Carpana, E., Marocchi, L., Gelmini, L., 1995. Evaluation of the API 50CHB system for identiWcation and biochemical characterization of Bacillus larvae. Apidologie 26, 11–16. Cornelis, G.R., Wolfwatz, H., 1997. The Yersinia Yop virulon: a bacterial system for subverting eukaryotic cells. Mol. Microbiol. 23, 861–867. Dancer, B.N., Chantawannakul, P., 1997. The proteases of American foulbrood scales. J. Invertebr. Pathol. 70, 79–87. de Graaf, D.C., Vandekerchove, D., Dobbelaere, W., Peeters, J.E., Jacobs, F.J., 2001. InXuence of the proximity of American foulbrood cases and apicultural management on the prevalence of Paenibacillus larvae spores in Belgian honey. Apidologie 32, 587–599. Dingman, D.W., Stahly, D.P., 1983. Medium promoting sporulation of Bacillus larvae and metabolism of medium components. Appl. Environ. Microbiol. 46, 860–869.

D.C. de Graaf et al. / Journal of Invertebrate Pathology 91 (2006) 115–123 Dobbelaere, W., de Graaf, D.C., Peeters, J.E., Jacobs, F.J., 2001a. Development of a fast and reliable diagnostic method for American foulbrood disease (Paenibacillus larvae subsp. larvae) using a 16S rRNA gene based PCR. Apidologie 32, 363–370. Dobbelaere, W., de Graaf, D.C., Peeters, J.E., Jacobs, F.J., 2001b. Comparison of two commercial kits for biochemical characterization of Paenibacillus larvae larvae in the diagnosis of AFB. J. Apicult. Res. 40, 37–40. Gordon, R.E., Haynes, W.C., Pang, H.N., 1973. The genus Bacillus. Agriculture handbook no. 427. U.S. Department of Agriculture, Washington, DC. Govan, V.A., Allsopp, M.H., Davison, S., 1999. A PCR detection method for rapid identiWcation of Paenibacillus larvae. Appl. Environ. Microbiol. 65, 2243–2245. Heyndrickx, M., Vandemeulebroecke, K., Hoste, B., Janssen, P., Kersters, K., De Vos, P., Logan, N.A., Ali, N., Berkeley, R.C.W., 1996. ReclassiWcation of Paenibacillus (formerly Bacillus) pulvifaciens (Nakamura, 1984) Ash et al., 1994, a later subjective synonym of Paenibacillus (formerly Bacillus) larvae (White, 1906) Ash et al. 1994, as a subspecies of P. larvae, with emended descriptions of P. larvae as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens. Int. J. Syst. Bacteriol. 46, 270– 279. Holst, E., Sturtevant, E.P., 1940. Relation of proteolytic enzymes to phase of life cycle of Bacillus larvae, and two new culture media for this organism. J. Bacteriol. 40, 723–731. Jarosz, J., Glinski, Z., 1990. Selective inhibition of cecropin-like activity of insect blood by protease from American foulbrood scales. J. Invertebr. Pathol. 56, 143–149. Katznelson, H., 1950. Bacillus pulvifaciens (n. sp.), an organism associated with powdery scale of honeybee larvae. J. Bacteriol. 59, 153–155. Kilwinski, J., Peters, M., Ashiralieva, A., Genersch, E., 2004. Proposal to reclassify Paenibacillus larvae subsp. pulvifaciens DSM 3615 (ATCC

123

49843) as Paenibacillus larvae subsp. larvae. Results of a comparative biochemical and genetic study. Vet. Microbiol. 104, 31–42. Lauro, F.M., Favaretto, M., Covolo, L., Rassu, M., Bertoloni, G., 2003. Rapid detection of Paenibacillus larvae from honey and hive samples with a novel nested PCR protocol. Int. J. Food Microbiol. 81, 195–201. Martirani, L., Varcamonti, M., Naclerio, G., De Felice, M., 2002. PuriWcation and partial characterization of bacillocin 490, a novel bacteriocin produced by a thermophilic strain of Bacillus licheniformis. Microb. Cell Factories 1, 1. Neuendorf, S., Hedtke, K., Tangen, G., Genersch, E., 2004. Biochemical characterization of diVerent genotypes of Paenibacillus larvae subsp. larvae, a honey bee bacterial pathogen. Microbiology 150, 2381–2390. Neumann, T., Reiche, R., Hentschel, E.J., Gräfe, U., 1997. Apidiothricin— a new chlorothricin-type antibiotic from Paenibacillus larvae larvae, the cause of American foulbrood. Apidologie 27, 177–179. Piccini, C., D’alessandro, B., Antunez, K., Zunino, P., 2002. Detection of Paenibacillus larvae subspecies larvae spores in naturally infected bee larvae and artiWcially contaminated honey by PCR. World J. Microbiol. Biotechnol. 18, 761–765. Pot, B., Vandamme, P., Kersters, K., 1994. Analysis of electrophoretic whole-organism protein Wngerprints. In: Goodfellow, M., O’Donnell, A.G. (Eds.), Chemical Methods in Prokaryotic Systematics. Wiley, Chichester, pp. 493–521. Prince, S., 2000. The basis of virulence of Paenibacillus larvae subsp. larvae, the causative agent of American foulbrood in honeybees, PhD thesis, School of Biosciences, University of Wales CardiV, CardiV, UK. Thorne, G.M., 1992. Diagnostic-tests in gastrointestinal infections. Curr. Opin. Gastroenterol. 8, 134–144. Vauterin, L., Vauterin, P., 1992. Computer aided objective comparison of electrophoretic pattern for grouping and identiWcation of microorganisms. Eur. Microbiol. 1, 37–41.