In-field Non-invasive Detection Of Honey Bee Pathogens

Background

The contribution of honeybees to agriculture indirectly through pollination services was estimated to be valued at around $19 billion in Australia in 2017, with the direct production of honey and other hive products accounting for approximately $100 million of this value. However, honeybee health has rapidly declined in recent years due to a variety of environmental stresses such as pathogens, agrochemical exposure, lack of quality forage, and reduced habitat.

Importance

Under current husbandry practice, opening beehives is required to assess bee health; however, this gives rise to additional stress to the bee’s which can take hours, or sometimes days, to full recovery that results in a negative effect on bee’s health and productivity.

In particular, this is a major concern as bees are generally asymptomatic when infected with viral pathogens however stress negatively impacts the immune system resulting in sudden deaths.

Benefits

The ability to detect pathogens by using a rapid non-invasive in-field method will be a major breakthrough in allowing beekeepers to limit stress to bees through a change in husbandry practice.

The assay has the potential to inform local treatment decisions for the management of bee health in real-time. This may help to identify infections before clinical symptoms occur, leading to early treatment and reduced spread, while simultaneously reducing stress in bees by eliminating the need to open hives to collect diagnostic samples.

We will utilize established methods in our laboratory using environmental sampling to develop LAMP assays to be able to detect Paenibacillus larvae and Melissococcus plutonius. This model will have several major outcomes and outputs.

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Outcomes

The key outcome for this project is the improved management of honey bee and hive health via the early detection of pathogens without the need to open the hive, causing undue stress to the bees. Once developed, this assay will be able to be performed in the field by people with minimal training which will lead to informed management decisions in real-time by apiarists. The knowledge gained from this project will have additional benefits in applying the technology to other honey bee pathogens, including pest insects such as the small hive beetle (Aethina tumida) or for floral identification.

Outputs

The proposed outputs of the research include:

• A molecular toolkit for sampling and detection of Paenibacillus larvae and Melissococcus plutonius without needing to open beehives
• An understanding of how to sample and detect other honey bee pathogens
• Publications in international research journals
• Generation of human capital with interest in beekeeping
• Development of hands-on training course in the use of diagnostic kits.

Project Design and Method – In 250 words or less describe the project design and method.

The development of non-invasive in-field rapid diagnostic for honey bee pathogens will occur in two stages over two years.

Stage 1(Year 1) We develop an environmental DNA sampling method based on using a range of different solutions that can extract and preserve the nucleic acid of the pathogen from swabbing the outside the hive box. A DNA plasmid containing a gene from Paenibacillus larvae or Melissococcus plutonius the causative agents of American and European foulbrood respectively will act as a control for development of the sampling method. Control plasmid will be placed on the outside of the hive boxes and swab will be taken to extract DNA.

Stage 2 (Year 2) Primer sets will be designed and optimized to amplify Paenibacillus larvae or Melissococcus plutonius DNA by use of loop-mediated isothermal amplification (LAMP). The specificity and sensitivity of the assay will be determined using a dilution of the control plasmids and suit of environmental bacterial DNA. The specificity and sensitivity LAMP assay will be also compared with the “gold standard” diagnostic test performed by qPCR

Field test swabs will be obtained, analysed by LAMP and compared with the qPCR test to determine is suitable to use as an in-field non-invasive diagnostic test. The potential for adoption and commercialization pathways identified. Adoption can be achieved by an energetic and innovative publication effort, or by the development of IP rights that provide their own commercialisation incentives to foster adoption. Reference to the Corporation’s commercialisation principles are contained on the IP & Commercialisation tab at http://www.rirdc.gov.au/for-researchers

The proposed outcomes of this project will be widely adopted by industry via the development of user-friendly training workshops on the use of the diagnostic kits. Traditional dissemination of results will be via publication in international journals, seminars at local, national and international meetings. In addition, social media, including websites, Wikipedia articles, twitter feeds, podcasts.

We also use an innovative type of publication in the Journal of Visualized Experiments which is a video-based scientific journal that allows the method to be shown rather read. This video will act as a ‘training’ video for the industry to perform the non-invasive sampling and detection of bee pathogens.

This project will form the basis of a rapid field diagnostic kit that will be manufactured by A/Prof. Beddoe’s industry partner Geneworks. The IP rights will be protected according to La Trobe’s University and AgriFutures policy.

The Animal BioSolutions laboratory based at the AgriBio Centre for AgriBioscience (La Trobe University) is a world leader for the development of in-field based diagnostic kits. We are currently the only research group in Australia that has direct commercial links to develop these in-field based diagnostic kits for other pathogenic diseases, with the expertise to validate diagnostics kits in the field.

Dr Timothy Cameron is an early career research scientist with a passion for agricultural animal disease management. He completed a PhD in 2018 involved in the development of a vaccine for a parasite that is a major problem in Australian livestock, liver fluke. Since working in CI Beddoe lab, he has expanded his interests to the diagnostics of pathogens and further vaccine development. Ultimately, Dr Cameron is aiming to develop his own research program in which honey bee health will play a major role. Dr Cameron will be the lead investigator on this project and be responsible for the day-to-day management of the project

Travis Beddoe is a mid-career research scientist that heads the Animal BioSolutions Laboratory at La Trobe University. He has extensive expertise in animal health management, in particular, boutique animal species such as crocodiles and racing pigeons. His research interests include field diagnostics, disease pathogenesis and vaccine development. He has had major success in translating his research findings to commercial outcomes. For example, he has licensed a vaccine for commercial production in racing pigeons, as well as forming a partnership with Geneworks to commercialize field-based diagnostics kits.