Progress report for GNC21-325
The health of honey bees is integral to sustainable crop production. European foulbrood (EFB) is a bacterial disease that severely affects the health of honey bees. For years, Michigan beekeepers have complained of high levels of this disease in their colonies. Our previous work shows that EFB is widespread in commercial colonies in Michigan, but many basic epidemiological questions regarding this pathogen remain unanswered. In 2019, in collaboration with the USDA ARS laboratory in Beltsville, MD, we were able to test samples from 78 honey bee colonies in Michigan managed by three commercial beekeepers. The purpose of this previous work was to validate a lateral flow test that can help diagnose EFB in the field. While we were able to confirm the sensitivity of the field test, there were multiple samples that showed a high bacterial load that tested negative by this device. Recent studies suggest that strain type may impact test results, but this has not been well studied, and nothing has yet been published on strain types in Michigan. Here we propose to use an established method for identifying strain types to identify the strains present in each of the 78 colonies. We hypothesize that the highly virulent atypical strain will be present in the false negatives, as there have been reports that this test may not be as sensitive in detecting these strains. We further hypothesize that multiple strains are circulating regionally and may be linked to travel. The results of this study will help us understand the impact strain type has on test results. Additionally it will give some basic information about what strain types are present among the sampled colonies. This is important as different strain types have been shown to have different levels of virulence for honey bees, and strain typing provides an important basis for future epidemiological study. Healthy honey bees are an important component of Michigan’s agriculture system, and understanding their diseases will benefit both beekeepers and the farmers that depend on them for pollination.
This project will have measurable impacts on multiple stakeholders. Beekeepers will, for the first time, know what strains of M. plutonius are present in Michigan. The 38 strains that have so far been identified vary widely in their levels of virulence, with some causing no observable disease and others causing extremely high colony mortality. This makes understanding what strains are circulating important in formulating and prioritizing response and treatment protocols.
Veterinarians will also benefit from this project. Recent regulations have required veterinary oversight in order for beekeepers to treat their hives. Many veterinarians are not experienced in diagnosing EFB, making rapid tests that can be deployed in the field an important part of disease management. The results of this study will help veterinarians and beekeepers better understand the results of this test. If a false negative is suspected, the results of this research can help explain why, and inform when treatment is necessary.
Apiary inspectors will also have additional insight into possible causes for varying disease prevalence and severity between and within operations. It will also give them tools to better understand between apiary transmission and insights into transmission that might be associated with out of state travel.
Finally, fruit and vegetable growers depend heavily on a healthy honey bee population for pollination services. Helping understand this disease is the first step to creating sustainable approaches to disease management that will positively impact honey bee health and increase the economic return of crops that depend on them for pollination.
1) Determine what strains of Melissococcus plutonius are present
Originally, we intended to perform multi-locus sequence typing (MLST) on 74 strains collected from two beekeeping operations in Michigan in order to identify regionally circulating strains. This procedure looks at small differences in four genes to determine what strain type is present. In the last two years the cost of higher definition analysis has reduced significantly, and we found that if we pooled our samples with those from other projects we could gain much better insight on the entire genetics of circulating strains from many operations. For that reason, a subset of samples from our 2019 project aimed at answering questions in objective two (the impact of strain type on diagnostics) were selected and will be submitted for multiplex sequencing with another broader surveillance project funded by the Rackham foundation and a longitudinal treatment trial funded by Project GREEEN. These two complimentary projects, in combination with resources provided by NC SARE will allow us to obtain whole genomic data bacterial strains from over 300 colonies from 15 beekeepers including commercial, sideliner and hobbiest and from three different states (Michigan, Georgia and California). This data will provide much more insight into circulating strains and the higher resolution will provide invaluable insight into investigations of transmission dynamics.
For that reason the supply budget for this part of the project was increased in order to accommodate the increased material needed for initial culture and isolation of these samples in preparation for full genetic sequencing. The revised budget allocates additional expenses for media gas mixtures needed for bacterial cultivation a well as disposables such as pipet tips and innoculation loops. The new budget was submitted and approved and most of the preparatory work has been completed well within the allocated budget.
2) Determine the role strain type plays on diagnostic results
Because we only had eight false negatives on the lateral flow device, we decided to focus on deeper sequencing of those strains and genetically comparing them to eight strains from ones that tested positive. The budget has been used to culture these samples on M110 agar under anaerobic conditions and isolate these cultures in KSBHI broth. A portion of this bacteria was saved for future studies and the remainder underwent DNA extraction and purification for whole genome sequencing. These samples are awaiting the final culture of samples collected in 2023 to submit for multiplexing. Analysis of this data to elucidate strains not detected by the LFD and their impact on hive health will be performed in the coming months.
3) Engage with stakeholders, including beekeepers and veterinarians about the results and what they mean for disease diagnosis and management
The current progress on this objective is outlined in the Outreach portion of this report and will continue to develop in the coming months as final results become available.
Educational & Outreach Activities
While our outreach on the major topics of this research is still forthcoming, we have been able to fulfill our goals of educating veterinarians on the methods used to diagnose honey bee diseases through multiple avenues including as part of a collaborating grant to train veterinarians. This work has been put into the form of a workshop presented to 25 veterinarians as part of the 2022 Honey Bee Veterinary Consortium which was hosted at Michigan State University August 27-28, 2022. Training took place in person to 25 veterinarians from around the country, and virtual training was also made available to a wider veterinary audience. Additionally, training wet labs were held with the MSU veterinary students as well as a day of diagnostics as part of the MSU veterinary clinical rotation. We estimate that these trainings have been made available to well over 200 veterinarians and veterinary students improving their ability and confidence in diagnosing this disease in honeybee colonies.
Materials are also being prepared for a wider audience of beekeepers and growers in the Heroes to Hives program and La Cosecha in the summer of 2023 and more details will be available as outcomes from our final report.
By providing veterinarians with the tools to diagnose EFB, we will provide a significant resource to beekeepers in need of treatment for their colonies. This disease causes a huge economic burden to beekeepers which is felt by every grower that depends on them for pollination services.
The primary focus of this project was to provide insights into an extremely widespread and poorly understood disease, European foulbrood (EFB), affecting honeybees and the crops that rely on them for pollination. Currently, very little is known about the strains circulating in Michigan. Our work to date has indicated that the bacteria that many colonies afflicted with this disease may be carrying multiple unique strains of the bacteria, each of which may play a unique role in disease progression and spread. Additionally, some of these strains may not be detected by a commonly used hive side test (lateral flow device). The final stage of our work is ongoing and this summer we will have whole genome data on circulating strains as well as strains that seem to evade detection by the diagnostic lateral flow device. Understanding these dynamics and educating the agriculture and veterinary community will be critical for improving diagnosis and treatment protocols that will improve the health of these important animals which are central to a healthy and sustainable agriculture system.