Final report for GNC21-325
Project Information
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. This project seeks to characterize circulating strains impacting of the pathogen using whole genome sequencing of isolates spanning five years collected from across the state. We hypothesize that the highly virulent atypical strain will be highly prevalent and associated with 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 which can help provide insights into transmission pathways. 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 operations. 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.
Cooperators
- (Researcher)
Research
1) Determine what strains of Melissococcus plutonius are present
Isolates of M. plutonius from 93 colonies spanning five years were selected to represent a broad range of Michigan beekeeping operations from across the state, including colonies during travel to California and Georgia. All 93 isolates were anaerobically cultured in KSBHI broth at 37C for 5 days and DNA was extracted using DNEasy Blood and Tissue kit following manufacturers instructions. Extracted DNA was submitted to MSU RTFS sequencing core for multiplex sequencing on Illumina Novaseq. Raw reads were uploaded to the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) [1] and reads were assembled de-novo using the Genome Assembly pipeline with Unicycler as the assembler. Draft assemblies were uploaded to PubMLST (https://pubmlst.org) for identity confirmation and strain type analysis. Additional phylogenetic analysis is ongoing.
1) Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Olson RD, Assaf R, Brettin T, Conrad N, Cucinell C, Davis JJ, Dempsey DM, Dickerman A, Dietrich EM, Kenyon RW, Kuscuoglu M, Lefkowitz EJ, Lu J, Machi D, Macken C, Mao C, Niewiadomska A, Nguyen M, Olsen GJ, Overbeek JC, Parrello B, Parrello V, Porter JS, Pusch GD, Shukla M, Singh I, Stewart L, Tan G, Thomas C, VanOeffelen M, Vonstein V, Wallace ZS, Warren AS, Wattam AR, Xia F, Yoo H, Zhang Y, Zmasek CM, Scheuermann RH, Stevens RL. Nucleic Acids Res. 2022 Nov 9:gkac1003. doi: 10.1093/nar/gkac1003. Epub ahead of print. PMID: 36350631 The Bacterial and Viral Bioinformatics Resource Center (BV-BRC), https://www.bv-brc.org/
2) Determine the role strain type plays on diagnostic results
In order to determine the impact of strain variation on diagnostic results, isolates from colonies that tested as false negatives on the lateral flow device and, for comparison, five isolates from colonies that tested positive were included in the whole genome sequencing and assembly from objective one. Identity and strain type was determined by uploading draft assemblies to PubMLST.
3) Engage with stakeholders, including beekeepers and veterinarians about the results and what they mean for disease diagnosis and management
Veterinarians
- Michigan State University Phi-Zeta day October 1st 2021 informing them on the reliability of field diagnostics for EFB.
- Diagnostics workshops were held for veterinarians as part of the 2023 Honey Bee Veterinary Consortium conference hosted at MSU on Friday, 8/28/2022
- Michigan Veterinary Medical Association conference 9/30/2023
Beekeepers
- Michigan’s commercial beekeepers at the Michigan Commercial Beekeepers meeting July 21st
- American Bee Research Conference – Thursday, January 11, 2024
- Instructional video on sample collection for EFB diagnosis for Heros to Hives virtual education has been filmed and is undergoing editing.
Growers
- Informational flyer was created for blueberry growers as part of La Cosecha program and translated into Spanish which will be distributed to growers and published online on MSU website.
1) Determine what strains of Melissococcus plutonius are present
Among 20 beekeepers spanning five years we determined four strains of Melissococcus plutonius are circulating in Michigan. The predominant strain was ST19, followed by ST12, both of which are considered highly virulent to honey bee larvae. Two other strains, ST39 and ST3 were also found in lower numbers. Whole genome sequencing revealed that none of these strains were unique, but whole genome data on two of the strains ST19 and ST3 have not yet been published and may yield important insights.
2) Determine the role strain type plays on diagnostic results
Screening by duplex PCR showed that that five of the 10 colonies had both strain groupings of M. plutonius present, typical and atypical; one of the five EFB positive LFD colonies and four of the five false negatives. When isolates were cultured, however, atypical M. plutonius was recovered from all colonies suggesting it was present but not detected by the PCR screening, and may have been at concentrations too low to interfere with the lateral flow device. Strain type recovered did not seem to affect LFD results and it is likely that in colonies with multiple strains, the amount of bacterium from each strain impacts test results. Future study using quantitative PCR on these isolates may help clarify this relationship.
|
Colony |
Duplex PCR Screen |
Cultured Sequence Type |
||
|
|
Typical |
Atypical |
||
|
Negative on LFD |
N1 |
X |
19 |
|
|
N2 |
X |
X |
19 |
|
|
N3 |
X |
X |
19 |
|
|
N4 |
X |
X |
12 |
|
|
N5 |
X |
X |
12 |
|
|
Positive on LFD |
P1 |
X |
X |
19 |
|
P2 |
X |
19 |
||
|
P3 |
X |
19 |
||
|
P4 |
X |
12 |
||
|
P5 |
X |
12 |
||
3) Engage with stakeholders, including beekeepers and veterinarians about the results and what they mean for disease diagnosis and management
Objective three results are outlined in the Education and Outreach section below.
Educational & Outreach Activities
Participation Summary:
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 in 2022 and 2023. Results were also presented at results at the Michigan Veterinary conference to an estimate 50 licensed veterinarians. We estimate that these trainings have been made available to well over 400 veterinarians and veterinary students improving their ability and confidence in diagnosing this disease in honeybee colonies.
Results were also shared with commercial beekeepers at the annual Michigan Commercial Beekeepers conference, July 21st, 2022 to approximately 40 commercial beekeepers. To reach additional beekeepers, a webinar was held as part of MSU extension outreach office hours on Nov. 9th, 2023 where the results were shared with an estimated 150 participants, and the recording was shared on the Michigan State University YouTube channel and has received over 1,000 views as of 3/24/2024. Results were also shared at the annual American Bee Research Conference in New Orleans on January 11, 2024 to an estimated 50 researchers and beekeepers.
Materials were 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. For Heroes to Hives, an instructional video is being prepared on sample collection to submit samples to the USDA for free disease diagnosis. Video has been shot for this portion but is still in the editing phase. When published it will be implemented into the Heroes to Hives curriculum giving participants the resources to determine if a bacterial disease is present in their colonies. This is expected to be finished later this year. For La Cosecha blueberry growers, a one sheet handout was prepared and translated into Spanish, explaining the disease and the seasonal impact it has on honey bee colonies.
By providing veterinarians and beekeepers 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.
Project Outcomes
Sustainable agriculture relies heavily on a healthy beekeeping industry to supply the pollination services needed. The bacterial disease European foulbrood presents a serious threat to honey bee health and a substantial economic burden on beekeepers. Additionally, veterinary oversight is needed to access the antibiotic needed to treat this disease, but veterinary training and diagnostic tools are lacking. This project makes a significant contribution to the sustainability of agriculture by providing additional resources and training to veterinary professionals about honey bee disease diagnostics. This training will help veterinarians make informed decisions about disease management. Beekeepers will benefit from increased access to diagnostics and agriculture as a whole will benefit from a healthier pollinator population. This project also provides the first substantial survey of strains circulating, providing a baseline for future epidemiological studies. Results will inform future control efforts, and genomic data will provide insights into the characteristics of the pathogen that causes this disease and possible pathways to mitigation.
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). Four primary strain types have been identified and whole genome analysis can help us better understand strain diversity and the impact the genes that might be driving virulence. Analysis of whole genome sequence data is ongoing, and we hope to have a manuscript published summer 2024 with our findings. 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.