Bacteriophage application in honeybees infected with Paenibacillus larvae 1st German Phage Symposium 9. 10. October 2017 University of Hohenheim LAVES Inst. f. Bienenkunde Celle 1
Paenibacillus larvae Rod-shaped entomopathogen Gram-positive, spore forming microaerophilic, endemic in honeybees Spores are the infectious agent (A. mellifera) only honeybee larvae can be infected Vegetative bacteria kill 100% of infected larvae larvae are killed by secondary metabolites LT 100 depends on the genotype Killed larvae are responsible for typical AFB-symptoms rotten smell heterogeneous brood areas only P. larvae can be isolated from remaining material LAVES Inst. f. Bienenkunde Celle 2
American Foulbrood (AFB) Larvae are infected with P. larvae-spores Spores germinate in the midgut bacteria proliferate massively host-killing by toxins Killed pupae are digested into glue-like liquid digestion by proteases P. larvae sporulates while drying out Nurse bees act as vector for spores LAVES Inst. f. Bienenkunde Celle 3
Apis mellifera and AFB Egg Lifestyle can be divided into two stages Brood (Egg, Larva, Pupa) Adult (Imago) [Larvae can be infected] [can act as vector for spores] Larva Pupa ERIC I ERIC II Larvae become infected after incorporation of spores Feeding from d 4 -d 9 Larvae can be infected in the first 36 hrs of live Infected larvae die after infection ERIC II: LT 100 ~ 7d ERIC II: LT 100 ~11d Dead larvae -become removed by nurse bees until d 9 Dead larvae -are lysed into glue-like liquid after closure of cells ( d 9 ) Adult LAVES Inst. f. Bienenkunde Celle 4
How to treat AFB? Burning colonies -killed colonies -financial losses +removal of contaminated material AFB agriculture.vic.gov.au Antibiotics (not allowed in Europe) -symptoms are masked -selection of resistances -residues in bee products infectious Shook-swarm method +adult bees keep alive +colonies need not to be killed -equipment has to be decontaminated LAVES Inst. f. Bienenkunde Celle 5
In vivo-exposure assays Preparing a colony Transfer of L1-larvae (d 4 ) Observe them for 14 d (d 18 ) Feeding different Diets (d 4-9 ) Daily inspection of individuals (d 4-18 ) Infecting larvae in the age 36 hrs (d 4 ) LT 100 (ERIC II) ~11 d (d 15 ) LT 100 (ERIC II) ~ 7 d (d 10 ) Single phage application ERIC I ERIC II during infection (d 4 ) Chronic phage application during feeding phase (d 4-9 ) LAVES Inst. f. Bienenkunde Celle 6
Single application in vivo-application of Phage HB10c2 48 larvae n = 3 t = 14 d t appl = 0 d (d 4 ) MOI Inf = 500 MOI Ther = 100 Results Control 25,0% ±2,1 Phage application 31,3% ±4,8[n.S.; p=2,99 x 10-1 ] C Infection ERIC I 100% [***; p=3,56 x 10-6 ] C Therapy ERIC I 99,3% ±0,7[n.S.; p=4,23 x 10-1 ] I Infection ERIC II 100% [***; p=3,56 x 10-6 ] C Therapy ERIC II 98,6% ±1,4[n.S.; p=6,77 x 10-1 ] II LAVES Inst. f. Bienenkunde Celle 7
Daily application in vivo-application of Phage HBχ 36 larvae n = 3 t = 14 d Δt appl = 1 d (d 4-9 ) MOI Inf = 500 MOI Ther = 100 Results Control 36,5% ±2,4 Phage application 27,3% ±1,8[ ** ; p=6,74 x 10-3 ] C Infection ERIC I 100% [***; p=4,08 x 10-9 ] C Therapy ERIC I 44,2% ±3,3[. ; p=7,49 x 10-2 ] C Infection ERIC II 100% [***; p=4,08 x 10-9 ] C Therapy ERIC II 35,5% ±6,4[n.S.; p=8,83 x 10-1 ] C LAVES Inst. f. Bienenkunde Celle 8
Summary American Foulbrood is caused by P. larvae devastating bee disease Efficient treatments are not known until now just spore reduction agriculture.vic.gov.au Different P. larvae-directed bacteriophages were isolated until now, all isolated phages are lysogenic In vivo-exposures suggest phagetherapy for AFB control daily application improves therapeutic effects LAVES Inst. f. Bienenkunde Celle 9
Acknowledgement LAVES Institut f. Bienenkunde Celle Werner von der Ohe TU Braunschweig, Inst. f. Mikrobiologie Michael Steinert AG Steinert, Gabi Günther Helmholtz-Zentrum f. Infektionsforschung Braunschweig Manfred Rohde Kathrin I. Mohr Leibnitz-Institut DSMZ Christine Rohde Johannes Wittmann Boyke Bunk Cathrin Spröer LAVES Inst. f. Bienenkunde Celle 10
Thank you, for your attention. 10/13/2017 LAVES Inst. f. Bienenkunde Celle Herzogin-Eleonore-Allee 5 29221 Celle 05141/90503-68 Hannes.Beims@LAVES.Niedersachsen.de 11