ApiLyse

Project Title
A novel bacteriophage-derived approach for Paenibacillus larvae control in Apiculture
Project Type
Nacional / Public
Funding Body
Funding Program
Projetos de Investigação Científica e Desenvolvimento Tecnológico - 2014
Reference
PTDC/CVT-EPI/4008/2014
Funding
  • CEB: 150 842,00
  • Total: 172 442,00
Start
01-07-2016
End
28-07-2019
Partnership
Universidade do Minho, Universidade de Évora

Principal Investigator

Team Members - CEB

Fellowship Members - CEB

Team Members - Non CEB

  • António Murilhas

Abstract

Honey bees are responsible for producing 1/3 of the world food supply by pollination of more than 100 species of flowering plants and the value of this pollination service is estimated at €153 billion annually. Honeybees are vital to the preservation and sustainability of terrestrial ecosystem, however; they are attacked by many pathogens as bacteria, fungi, viruses and parasites. One of the most devastating bacterial diseases affecting honeybee larvae is American Foulbrood (AFB), caused by Paenibacillus larvae, a worldwide-distributed spore forming gram-positive bacterium that spreads easily across apiaries producing highly resistant spores. When beekeepers suspect of AFB, complete burning of contaminated hives is the appropriated measure, which cause serious economic losses. Although antibiotics could be an option for the treatment of AFB, the consequent problems with bacterial resistances and the strict European legislations prohibiting antibiotics in honey for human consumption, discard this treatment choice. In this context, ApiLyse aims to demonstrate that bacteriophage (phage)-derived products are natural, reliable and valuable antimicrobial tools to control AFB. Phages are ubiquitous in nature, showing no toxicity to animals, plants or humans, and they specifically recognize their host bacteria, infect and multiplying inside these hosts. Phages produce enzymes called endolysins, that breakdown the host cell wall releasing progeny that start new infection cycles. Although endolysins alone are highly explored in medical fields, for their rapid killing of gram-positive bacteria within due to osmotic lysis, this approach in apiculture it totally new. ApiLyse aims to provide a proof-of-concept of the efficacy of phages and endolysins to control P. larvae inside honeybee larvae. We hypothesize that upon ingestion of the phage-based product developed by larvae, the bacteria will be destroyed in the gastro-intestinal tract, eradicating its capacity to cause clinically relevant symptoms. To accomplish ApiLyse’s aims, it will be necessary to firstly isolate and characterize P. larvae strains from Portuguese and European apiaries exhibiting the disease enabling an expressive epidemiologic study of the incidence of AFB in Portugal and an indicative study of the European situation relatively to this disease. Despite the 5 P. larvae phages already isolated and deposited in the CEB’s collection (genome sequence of one already deposited in the GenBank (KF010834)), the next task involves isolation, and characterization of more phages also from European samples. Genome annotation will identify putative lysins that will be cloned and heterologously expressed as already demonstrated with PlyPl23. The in vitro efficacy evaluation of this endolysin envisages promising results to in vivo P. larvae control. Since no toxicity signs were observed in artificially raised larvae when ingesting one of the phages (vb16) or the lysin PlyPl23, the assessment of the lytic efficacy of new phages and lysins against different P. larvae serotypes will be the following in vivo experiments. Actually, the most promising phages and lysins comprising the product formulation will be tested in vivo, at lab- and field scales. The scale up of the production of phage(s) and endolysin(s) will be optimized in fermenters. ApiLyse comprises an interdisciplinary research team, combining extensive microbiology, phage biology/therapy and protein engineering experience [Center of Biological Engineering (CEB), University of Minho (UM)], with know-how in apidology research [Department of Animal Production (DAP), University of Évora (UE)]. The group also has skills in modeling population dynamics in fermenters. CEB/UM main research has broad competence in the isolation, description and evaluation of the phages, annotation of phage genomes, cloning of interesting lytic enzymes, scale up the production of the phages/enzymes that reveal higher efficacy. DAP/UE is experienced in bee pathology applied research, and honeybee breeding. This institution counts with important networks in apidology, including participation in EU-funded projects, e.g. COLOSS. DAP/UE has also straight connections with Portuguese veterinary authorities (DGV) that will facilitate collection of samples from apiaries across central and southern Portugal, and also with several European institutions that can supply P. larvae contaminated samples. DAP/UE is highly skilled in supervision of the in vivo experimental work involving honeybee individuals/colonies. Besides this team, a skillful beekeeper, Zootechnical Engineer will be hired as a consultant for the practical implementation of the in vivo testing section of the project. CEB/UM counts with researchers allowed by FELASA to perform animal experiments.