General enquiries on this form should be made to:sciencesearch.defra.gov.uk/Document.aspx?Document=PH0502... · Web viewAs with other forms of livestock, honey bees are subject to

General enquiries on this form should be made to:Defra, Science Directorate, Management Support and Finance Team,Telephone No. 020 7238 1612E-mail: [email protected]

SID 5 Research Project Final Report

SID 5 (Rev. 3/06) Page 1 of 16

NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code PH0502

2. Project title

Assessing the effectiveness of shook swarm as a husbandry method for the control of European foul brood

3. Contractororganisation(s)

National Bee UnitCentral Science LaboratorySand HuttonYorkYO41 1LZ

54. Total Defra project costs £ 185,393(agreed fixed price)

5. Project: start date................ 22 May 2006

end date................. 30 December 2008


6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.European foulbrood (EFB), caused by the bacterium Melissococcus plutonius (ex. White, 1912), is one of the most serious, infectious and economically important brood diseases known to affect the honey bee (Apis mellifera). EFB occurs on every continent where apiculture is practiced, and it is the most widespread bacterial brood disease in Great Britain. The total number of English and Welsh colonies found to be infected between 2005 and 2007 was in excess of 500 per annum. The primary aim of this project was to investigate the field-scale efficacy of the available control methods for EFB. These include antibiotic application, and husbandry methods applied both at the level of the diseased colony and the whole apiary. The secondary aim was to obtain a better understanding of EFB epidemiology by collecting data regarding the national and within-hive distributions of the causative organism M. plutonius, as well as other bacteria associated with the disease. Project steer was provided by meetings of the Bee Health Advisory Panel which met four times during the project.

Two field trials were conducted to establish which control method(s) was/were superior for the management of EFB in A. mellifera: The first trial compared the efficacy of the standard available antibiotic treatment for EFB, oxytetracycline (OTC), with the success of a husbandry-based approach known as shook swarm (SS). SS is a non-chemical approach which involves transferring all adult bees from an infected hive into a clean hive, thereby removing the infected larvae from the colony. The second trial compared SS of symptomatic colonies against SS of all the colonies in the affected apiary (whole apiary SS). Treatment methods were evaluated by monitoring several criteria: First, samples of adult bees and larvae were collected, the genetic material extracted and tested for the presence of M. plutonius; second, the amount of M. plutonius was determined using quantitative, species-specific real-time PCR (qPCR). Finally, disease occurrence and colony mortality rates were monitored. Significant differences were observed in the likelihood of treated colonies to test positive for M. plutonius using real-time PCR, depending on the treatment method employed. For the first trial in the season post-treatment, OTC-treated colonies were more likely to test positive for M. plutonius than SS-treated colonies. For the second trial in the season post-treatment, colonies from apiaries treated using SS were more likely to test positive for M. plutonius than colonies from apiaries treated using the SS whole apiary approach. Interestingly, contact colonies (i.e. asymptomatic colonies in diseased apiaries) were more likely to test positive than asymptomatic colonies from disease free apiaries, suggesting an inoculum burden on larvae and adult bees in the contact colonies. The levels of M. plutonius were not significantly different between either OTC and SS treated colonies (first trial) or SS treated colonies and the whole apiary SS approach (second trial) when measured after treatment with qPCR. Significant differences were observed in the amount of M. plutonius between symptomatic and asymptomatic colonies. These differences were significant for both larvae and adult bee samples using qPCR (p < 0.001). Disease reoccurrence was measured as EFB presence between the time of treatment and the end of the season following treatment.


Colonies treated in the first trial incurred disease reoccurrence more frequently after treatment with OTC (22%) compared to SS (4%) (p=0.037). Colonies treated in the second trial incurred disease reoccurrence more frequently in apiaries where only symptomatic colonies were treated using SS (17%) compared to apiaries where SS was applied to the entire apiary (8%), although this did not reach statistical significance (p=0.16). Clearly a large year-to-year variation in the efficacy of SS treatment was observed between the first and second trial. However, the higher reoccurrence in the second trial occurred in a year which showed a 34% increase in national EFB incidence compared to the first year. High levels of colony mortality were experienced in both years, however, this did not differ significantly between colonies receiving treatment and untreated control colonies in either year (2006 p=0.77; 2007 p=0.88).

Over 80 samples from apiaries in the north of England and north Wales tested negative for M. plutonius. Samples from only one northern apiary tested positive for M. plutonius, suggesting the bacterium is not distributed widely in Northern areas. There were no differences in the amount of M. plutonius in foraging versus hive bees using qPCR, suggesting infected foragers may post a high risk of colony-colony spread. Dissections revealed the distribution of inoculum in adult bees from EFB-infected colonies, suggesting all external and alimentary organs were positive for M. plutonius. Interestingly the bacterium was most prevalent in the rectum of the honey bee, an area known to contain a high titre of anaerobic organisms.

The main findings of this project have been effectively disseminated by way of presentations, paper summaries for beekeepers, training by bee inspectors and the submission of a manuscript for peer-review publication. The uptake of husbandry-based methods of EFB-control have increased since this project was commissioned, demonstrating effective extension of research findings to beekeepers. The data generated in this project will assist with improved, evidence-based management of EFB in England and Wales.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Introduction

There are several hundred thousand honey bee (Apis mellifera) colonies across the UK. Some 250,000 colonies of these are managed by 37,000 beekeepers in England and Wales. Around 300 beekeepers manage bees on a professional basis and are members of the Bee Farmers’ Association; collectively, they manage about 40,000 colonies; the remainder are small scale producers. UK honey production is worth in excess of £20m p.a, but of far greater economic significance, is the service provided by bees as primary pollinators: the value of commercial crops grown in the UK that benefit from bee pollination is ~£200m p.a. (Carreck & Williams, 1998), and A. mellifera is also a crucial pollinator of many species of wild flora. Internationally, the honey industry produces some 1,381,000 tons (worth £7.5 b.p.a. at UK prices) and pollination services are estimated at ~£70b p.a.) (Borneck & Merle, 1989; Corbet et al., 1991; Morse & Calderone, 2000; Gordon & Davis, 2003). Bee health and colony declines are now matters of major international concern, not only to beekeepers, but also to food producers (who rely on the pollination services of bees), the scientific community, and to the broader public. There is a clear need to maintain healthy UK honey bee stocks, and to protect them against infection and/or infestation by the various pests and pathogens which may affect them.

As with other forms of livestock, honey bees are subject to a range of harmful diseases. Some of these affect the adult bees; others affect the immature stages of bees’ development (larvae and pupae) and these are referred to as brood diseases. One of these is the serious, infectious and economically important brood disease known as European foul brood (EFB), caused by the bacterium Melissococcus plutonius (ex White 1912) (Aleksandrova, 1949; Bailey & Collins 1982). EFB occurs on every continent where apiculture is practiced


(Matheson, 1993), and it is the most widespread bacterial brood disease in Great Britain. The total number of English or Welsh colonies found to be infected between 2005 and 2007 was in excess of 500p.a. (Wilkins et al., 2007). M. plutonius multiplies in the mid-gut of infected larvae, competing with them for their food resources (Bailey, 1960; 1983; Bailey & Locher, 1968; Shimanuki, 1997, and references cited therein). Larvae usually die within 1-2 days before being sealed in their cells, or sometimes shortly afterwards, but always before pupation. Signs of EFB include brood displacement in cells, yellow larvae, and larvae that become flaccid and decompose (Bailey, 1961). Some infected larvae are able to survive, if they receive a sufficient abundance of food to enable them to complete their development whilst supporting their bacterial load. However, when such infected survivors pupate, they void their gut contents into the cell, contaminating the comb with millions of infective bacteria. Eventually the bacteria infect such a high proportion of the brood that the colony will be weakened, and ultimately killed. EFB is a notifiable disease under the Bee Diseases and Pests Control (England or Wales) Orders 2006, and is therefore subject to official control by the examination of colonies for signs of the disease, and compulsory treatment or destruction of diseased colonies.

Currently there are several options for the control of EFB employed by the NBU Bee Inspectorate: If disease levels are perceived as low, applications of an antibiotic, Oxytetracycline (OTC), are known to assist colony recovery (Thompson & Brown, 2001). Alternatively, in cases of higher disease levels where the only course of action is destruction, colonies are burned. A third option, however, is a husbandry technique known as Shook Swarm (SS) (Waite et al., 2003). SS has been developed as an alternative to the use of antibiotics to achieve improved disease control of EFB. It involves transferring all adult bees from an infected hive into a clean hive, thereby removing the infected brood from the colony. Provisional results have shown that SS can offer higher levels of success, with lower levels of disease recurrence, than OTC application. SS removes the primary inoculum source from the colony, rather than simply suppressing the bacterial load to sub-clinical levels. However, insufficient data has been available on bacterial incidence to allow SS to be recommended as an effective alternative to antibiotics for the treatment of EFB. In additional, our understanding of EFB epidemiology and M. plutonius distribution is poor.

Aims and Objectives

The primary aim of this project was to investigate the field-scale efficacy of the available control methods for EFB. These include antibiotic application, and husbandry methods applied at either the level of the diseased colony or that of the whole apiary. The secondary aim was to obtain a better understanding of EFB epidemiology by generating data looking at national and within hive distribution of the causative organism, as well as other bacteria associated with the disease. Specifically, the objectives of this project were to:

2006O1: Convene steering group meetings O2: Identify and select suitable apiaries O3: Apply disease controls and conduct sampling O4: Analyse samplesO5: Undertake follow up assessments of coloniesO6: Disseminate information

2007O7: Assess efficacy of whole apiary approachO8: Undertake cost benefit analysis of SS techniqueO9: Investigate the role of adult bees and secondary bacteria in the epidemiology of EFB O10: Investigate the epidemiology of M. plutonius in adult honey bees

Methods

Steering group meetings (O1)Meetings of the Bee Health Advisory Panel were used to discuss project progress. These meetings involved industry contacts from key beekeeping organisations (WBKA, BFA, BBKA, CONBA, BIBBA, BDI) and individuals from Defra Plant and Bee Health Division.

2006 field trial (O1-5)The first year field trial was designed to monitor the bacterial loading, mortality and ultimately disease reoccurrence in EFB affected colonies treated with either SS or OTC applications, respectively. It also sought to monitor “contact colonies”, which were asymptomatic of disease but were located in apiaries where EFB was known to be present. Additional data was also gathered to compare levels of M. plutonius measured in contact colonies with those found in asymptomatic colonies where disease was absent. The number of combs of larvae and adult bees present in each colony was used as a measure of colony health. Both measures were included as


explanatory variables in all subsequent analyses. Colonies where these data were not collected were excluded from the analyses.

In 2006 and early 2007, samples of both adult bees and larvae were collected from EFB-infected and EFB-free apiaries throughout England and Wales. NBU inspectors selected appropriate colonies from apiaries for inclusion in this project during statutory disease inspection programmes. All inspections were carried out to NBU Good laboratory Practice (GLP) standard operating procedures. For the purposes of this experiment, a total of 169 colonies were included in the sampling regime. Bulk samples of adult bees and brood (n=100) were taken from the brood nest area of infected and healthy colonies. Colonies symptomatic for EFB were selected randomly for either SS (n=20) or OTC (n=26) treatment. Shook swarm treatment consisted of removal of adult bees to a clean hive containing new foundation followed by feeding as required. OTC treatment consisted of a single dose of antibiotic (1 g ai) in 250 ml sucrose solution (1 kg sucrose in 568 ml water) trickled onto cells of an empty frame on the edge of the brood nest. Samples were also collected from contact colonies (n=48) and symptomless colonies from apiaries that were free from EFB (n=59).

For each colony, samples of larvae and adult bees were collected in 35 ml of collection buffer at four time-points: pre-treatment (A); 1-2 months post-treatment (B); end of the 2006 season (C); and in spring 2007, with an incomplete replication of colonies for each time-point. A DNA extract was prepared from approximately 100 individuals from each sample by grinding to homogeneity in a ‘long special universal’ bag using a Lenze grinder (Bioreba). DNA was extracted from cleared lysate using a Wizard® Magnetic DNA Purification System for Food (Promega, FF3750) in conjunction with a Kingfisher ML magnetic particle processor (Thermo Electron Corporation) as described by Ward et al. (2007). Once complete, all extracts were stored at –80°C prior to analysis.

Each DNA extract was tested for the presence of M. plutonius and A. mellifera using real-time PCR. Real-time primers and probe were designed using Primer Express software (Applied Biosystems, Branchburg, New Jersey, USA). EFB assay EFBFor/EFBRev2/EFBProbe was selected to amplify a specific fragment of the 16S rRNA from M. plutonius (Accession AJ301842). The closest sequence matches for each primer were determined by comparing all the published sequences on the NCBI database using the BLASTN search algorithm. Assay specificity was confirmed by testing against nucleic extracts from a panel of bacteria to have a known association with honey bees or EFB. Assay AJ307465-955F/1016R/975T was designed to A. mellifera 18S rRNA (Ward et al., 2007), and used as an internal control to normalise for extraction efficiency. Real-time PCR reactions were set up as duplicate 25 μl reactions using Applied Biosystems TaqMan® reagents (product number 4304441) following manufacturer’s protocols. The reaction mixture consisted of 1X TaqMan Buffer A, 5.5 mmol-1 MgCl2, 200 nmol-1 each dATP, dCTP, dGTP, and dTTP, 0.625 i.u. AmpliTaq Gold DNA polymerase, 300 nmol-1 reverse and forward primers, 100 nmol -1 probe, and 10 μl template in 25 μl volume. TaqMan® PCR reactions were carried out within the ABI Prism 7900HT Sequence Detector System (PE Biosystems) beginning with 50˚C for 2 min, 95 ˚C for 10 min, and 40 cycles of 95 ˚C for 15 s and 60 ˚C for 1 min. Quantification of M. plutonius in each sample was achieved using the method described by Gil-Salas et al. (2007) with assay EFBFor/EFBRev2/EFBProbe as the target and assay AJ307465-955F/1016R/975T as the reference assay. For the target assay, a 1:10 dilution series was created by adding 10 to 1 x 10 8 of M. plutonius cells (Type strain LMG 20360) to crushed healthy A. mellifera. An extract containing 2.5 ng/ul of healthy A. mellifera DNA (equating to 9,000 copies of the haploid honey bee genome) was used to create the dilution series for the reference assay. The final calculation for the actual quantification of each DNA extract was expressed as the number of cells of M. plutonius per haploid honey bee cell.

Quantitative real-time PCR data were analysed using a linear model with log-normalised quantity of M. plutonius DNA as the response, and treatment and sample timing as the predictors. Qualitative data were analysed using logistic regression with PCR result as the response variable (expressed as binary with C T 40 as the threshold) and treatment as the predictor. Differences between post-treatment samples were investigated, and where appropriate pre-treatment (timing A) versus post-treatment (timing B,C,D) comparisons were made. Reoccurrence of disease on treated colonies and colony loss figures were collated from inspection data at the end of the 2007 inspection season and analysed using logistic regressions. Means were separated using least significant difference as appropriate. All analyses were completed using Genstat version 11.1 or Graphpad Prism version 3.02.

2007 field trial (O7)The second year field trial was designed to compare the bacterial loading, mortality and disease occurrence in apiaries where EFB symptomatic colonies were treated individually with SS, with that recorded in diseased apiaries where all colonies were treated with SS irrespective of whether they were symptomatic (i.e a whole apiary approach was adopted). Apiaries were carefully selected by the NBU Bee Inspectorate. Where possible, two apiaries owned by the same beekeeper were chosen to represent each treatment. In total, 16 apiaries were selected with an even split between SS treatment of symptomatic only and whole apiary approaches (n=8). Samples of adult bees and brood were collected from diseased and contact colonies (SS symptomatic 46 colonies; whole apiary 34 colonies). Samples were collected on four separate occasions: A) Prior to treatment; B) 6 weeks post SS; C) at the end of the 2007 beekeeping season; and D) Spring 2008. Sampling, real-time PCR, quantification, disease occurrence rates and statistical analyses were all completed as described for the 2006 field trial. The number of combs of larvae and adult bees present in each colony was taken as a measure of


colony health. Both measures were included as explanatory variables in all subsequent analyses. Colonies where these data were not collected were excluded from the analyses.

Cost benefit analysis (O8)Beekeepers included in the trial were provided with questionnaires to help determine the actual cost of SS, including frames, foundation, and change in income due to increases or decreases in honey productivity. In addition, average prices of combs and foundation were collated from two leading UK retailers (Thornes and National Bee supplies) and frame replacement of a single brood box for National and Commercial hives compared to the cost of OTC application.

Investigations into the epidemiology of EFB (O9-10)Four experiments were completed to investigate the epidemiology of EFB. Firstly, in order to investigate the risk of foraging bees transferring the bacterium between hives, the amount of M. plutonius was quantified in 5 samples of foraging bees and compared to the amount found in hive bees from the same colonies. Secondly, individual adult bees (n=6) were dissected from a sample testing positive for EFB, and specific body parts (crop and ventricles, head, legs and wings, rectum, thorax, venom bulb, and venom sac respectively) screened for the presence of M. plutonius. Thirdly, samples (n=64) were collected from a new outbreak in Cumbria to monitor the speed of spread of M. plutonius into the local environment. Fourthly, asymptomatic and symptomatic larvae were sampled from diseased combs and subjected to pyrosequencing to determine the presence, and therefore potential role, of any secondary bacteria. The latter experiment is included in the final report for project PH0505.

Results

Steering group meetings (O1)Meetings of the Bee Health Advisory panel were held 4 times during the course of the project (3rd April 2007, 30th

October 2007, 11th March 2008, 8th April 2008). Meetings were attended by key industry contacts, staff from Defra policy, members of the National Bee Unit and other scientists from Fera. Meetings provided effective project steer and a platform for initial sharing of results with the honey beekeeping industry.

2006 field trial (O2-5)Real-time PCR assay EFBFor , EFBRev2 and EFBProbe was designed to specifically detect M. plutonius 16S rRNA. Results of the BLASTN searches revealed no exact nucleotide matches for either primer sequences other than four sequences of M. plutonius 16S rRNA (AJ301842, AY862507, X75751, X75752). Assay EFBF/EFBR/EFBProbe only reacted with DNA preparations of M. plutonius and showed no cross-reaction with a panel of other bacteria (Table 1).

In total, 367 adult bee and 352 larvae samples were included in the analysis. Timing varied slightly between SS (09/06/2006-11/08/2006) and OTC (12/06/2006-21/09/2006) treatments (Table 2). There was a significant association between visual EFB symptoms and the real-time PCR result for samples collected pre-treatment (p < 0.001). All larvae (46/46) and most adult bees (45/46) collected pre-treatment from colonies with EFB symptoms tested positive for M. plutonius using real-time PCR. Although the majority of larvae and adult bee samples from all symptomatic colonies tested positive for M. plutonius prior to treatment, the proportion of positive samples dropped after Shook swarm or OTC treatments had been applied (Table 2).

At sampling point A (Summer 2006), a significantly higher proportion of larvae and adult bees samples from asymptomatic contact colonies tested positive for M. plutonius compared to asymptomatic colonies sampled from EFB free apiaries (Larvae p=0.02; Adult bees p<0.001) (Figure 1A). Logistic regression analysis of samples of larvae and adult bees collected post treatment (timing B, C, D) did not show any significant difference in the proportion of positive data between sampling times (Larvae p=0.26; Adult bees p=0.17), so these data were grouped to examine the pooled post treatment proportion positive data. These data demonstrated significant differences between groups in the proportion of samples that tested positive for M. plutonius (Larvae p=0.001; Adult bees p<0.001). A mean separation grouping showed that fewer SS-treated and contact colonies tested positive for M. plutonius compared to those treated using OTC (Figure 1B).

Table 1. Specificity testing of real-time assay against DNA preparations from a panel of bacteria.

Species CSL number LMG number Average CT

Paenibacillus larvae 6254 9820 40.00P. larvae 6255 14425 40.00P. larvae 6256 14426 40.00


P. larvae 6257 16147 40.00P. larvae 6258 16148 40.00P. larvae 6259 16241 40.00P. larvae 6260 16250 40.00P. larvae 6261 16251 40.00P. larvae 6262 14428 40.00P. larvae 6263 15974 40.00P. larvae 6264 16247 40.00P. larvae 6265 16249 40.00P. larvae 6266 16252 40.00Paenibacillus alvei 6558 13255 40.00P. alvei 6559 13260 40.00P. alvei 6560 16913 40.00P. alvei 6561 17051 40.00P. alvei 6562 17052 40.00P. macerans 6563 6324 40.00P. macquariensis 6564 6935 40.00P. polymyxa 6565 13924 40.00Achromobacter eurydice 7100 - 40.00Brevibacillus laterosporus 6672 16000 40.00B. laterosporus 6673 15110 40.00Enterococcus faecalis 6674 7937 40.00Melissococcus plutonius, Type strain 6679 20360 27.78M. plutonius, UK 7086 - 23.52M. plutonius, UK 7087 - 21.98M. plutonius, Thailand 7148 - 21.45M. plutonius, Thailand 7149 - 21.62Water control - - 40.00Capped water control - - 40.00

Table 2. Real-time PCR results for samples of larvae and adult bees (in parentheses) for each treatment by sample timing for 2006 field trial.

Sample timing

Shook swarm OTC Contact colony Healthy apiaryPCR

positivePCR

negativePCR

positivePCR

negativePCR

positivePCR

negativePCR

positivePCR

negative

Pre-treatment A 20 (20) 0 (0) 26 (25) 0 (1) 22 (19) 24 (29) 15 (3) 44 (56)

1-2 months post-treatment B 5 (3) 18 (20) 18 (13) 10 (16) 9 (6) 20 (27) - -

End of 2006 C 8 (3) 6 (11) 7 (4) 5 (8) 5 (2) 6 (12) - -

Spring 2007 D 3 (3) 14 (14) 14 (14) 9 (13) 19 (13) 25 (32) - -


Figure 1. Estimated proportion of asymptomatic colonies that tested positive for M. plutonius at timing point A (A) and post- treatment (B) using real-time PCR. Means were separated using

pairwise testing at p=0.05 (different suffix [a or b] represents a significant difference). Error bars represent 95% confidence intervals.

Standard curves prepared from serial dilutions of M. plutonius and honey bee DNA were suitable for quantification with respective R² values all above 0.99 (Data not shown). A linear regression analysis of log quantitative data collected post treatment (timings B, C, D), using only PCR positive data, did not show any significant differences in the amount of M. plutonius between sampling times (Larvae p=0.82; Adult bees p=0.08). These data were therefore grouped to examine the pooled post treatment amount of M. plutonius. When the post treatment data were combined, regression revealed significantly higher levels of bacteria in larvae and adult bees from symptomatic colonies compared to those found in similar individuals collected from asymptomatic colonies before treatment in 2006 (Figure 2). Shook swarm and OTC treatments each significantly reduced the levels of bacteria in larvae after treatment (Figure 2A). Reductions were also observed in the number of bacteria in adult samples, however this reduction did not achieve significance for SS treated colonies (Figure 2B). In addition, both treatments reduced the levels of M. plutonius bacteria in larvae and in adult bee samples collected post-treatment (timing B, C, D) to similar levels as colonies that had been asymptomatic in 2006 (Figures 2A & 2B).

Figure 2. Quantitative real-time PCR result for samples of larvae (A) and adult bees (B) collected pre-treatment (timing A); and post-treatment (timings B,C,D). The log number of M. plutonius

bacteria was normalised against the number of copies of the honey bee genome in each sample. Means represent predictions from a linear regression. Means were separated using least significant

differences after adjusting for multiple comparisons within sample timing.

Logistic regression analysis of EFB reoccurrence in treated symptomatic colonies suggested a borderline significance when SS treated, OTC treated and untreated colonies were compared (Change of deviance = 4.33, df = 1, p=0.037). However, the low number of observations in some categories may invalidate some of the


assumptions of this model, suggesting these results should be interpreted with care. The probability of EFB reoccurrence was lower in SS treated colonies (0.04) compared to OTC treated colonies (0.22). In 2007, no significant differences in colony mortality were recorded between SS treated (6/22), OTC treated (9/36) and untreated colonies (11/54) (Chi-square = 0.51, df = 2, p = 0.77).

2007 field trial (O7)In total, 34 symptomatic colonies and 46 asymptomatic colonies were repeatedly sampled across 16 apiaries, totalling 466 individual samples. All samples of larvae and adult bees collected from symptomatic colonies pre-treatment tested positive for M. plutonius using real-time PCR, and a significant association between visual EFB symptoms and the real-time PCR result was demonstrated (p < 0.001). The proportion of positive samples dropped after treatment for both SS and whole apiary treatments (Table 3).

Table 3. Real-time PCR results for samples of larvae and adult bees (in parentheses) for each treatment by sample

timing for 2007 field trial.

Sample timingShook swarm Whole apiary

PCR positive

PCR negative

PCR positive

PCR negative

Pre-treatment 34 (33) 9 (13) 25 (23) 6 (11)1-2 months post-treatment 15 (18) 16 (22) 10 (9) 23 (28)End of 2007 16 (15) 13 (15) 3 (5) 20 (20)Spring 2008 4 (7) 11 (13) 3 (4) 9 (13)

Linear regression analysis of log quantitative data on real-time PCR positive samples demonstrated a significantly higher amount of M. plutonius in larvae and adult bees from symptomatic than asymptomatic colonies (p < 0.001; Figure 3). Whilst colonies from the whole apiary treatment that tested positive for M. plutonius contained less bacteria than those treated using symptomatic SS, this did not reach statistical significance for samples of larvae (p=0.21) or adult bees (p=0.62) (Figure 4).

Figure 3. Quantitative real-time PCR result for samples of larvae (A) and adult bees (B) collected pre-treatment (A) from symptomatic and asymptomatic colonies. The log number of M. plutonius bacteria was normalised against the number of copies of the honey bee genome in each sample.

Means represent predictions from a linear regression. Error bars represent 95% confidence intervals.

SID 5 (Rev. 3/06) Page 10 of 16

Figure 4. Quantitative real-time PCR data for M. plutonius positive samples of larvae (A) and adult bees (B) treated using symptomatic SS or whole apiary SS. Post-treatment observations pooled from

timing B,C and D. Means represent predictions from a linear regression. Error bars represent 95% confidence intervals.

Logistic regression analyses revealed no evidence of any difference in the proportion of colonies testing positive for the three post-treatment observations (timings B, C, D; Larvae p=0.17; Adult bees p=0.72), but there was an effect of treatment (Larvae p=0.004; Adult bees p=0.003). As a result, all three post-treatment observations were combined in a further logistic regression that demonstrated strong evidence of a difference between pre and post-treatment observations (Larvae and Adult bees p<0.001), as well as an effect of treatment (Larvae p=0.02; Adult bees p=0.01). Pairwise tests confirmed no significant difference in the proportion of colonies testing positive for M. plutonius pre-treatment. However, post-treatment colonies from the whole apiary SS treatment showed a significantly lower probability of testing positive using real-time PCR than colonies from the symptomatic SS treatment (Figure 5).

Figure 5. Estimated proportion of colonies that tested positive for M. plutonius by real-time PCR when sampling larvae (A) or adult bees (B). Means were separated using pairwise testing at p=0.05.

Error bars represent 95% confidence intervals.

Logistic regression analysis suggested no significant difference in EFB occurrence in 2008 between the two SS treatments (p = 0.16; figure 6A). Whilst colony mortality was high during the trial, no significant differences in colony mortality were recorded between the two SS treatments and untreated EFB-free samples collected in Cumbria (Chi-square, p = 0.88; figure 6B).

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Figure 6. Re Occurrence of EFB in treated apiaries (A) and mortality (B) for the trial conducted in 2007. Error bars represent 95% confidence intervals.

Cost benefit analysis (O8)Initially, all beekeepers involved in the trial agreed to complete a questionnaire allowing the cost benefit analysis of EFB treatment. However, despite repeated requests, only 4 of 12 partially completed questionnaires were returned. These data indicate losses in honey income varied were £8 or less per shaken colony. SS requires additional feeding which cost between £2.50 and £3.82 per shaken colony depending on the apiary. The cost of OTC provision in 2007 was £12 per colony compared to an average cost for ready-made replacement frames to fill a single brood box of £37 (National hive) to £50 (Commercial hive).

Investigations into the epidemiology of EFB (O9-10)The amount of M. plutonius was quantified on bulk samples (n=100) of foraging bees and hive bees from the same colonies (n=5). All samples of foraging bees (average CTs 22.52 to 29.22) and hive bees (average CTs 21.59 to 27.03) tested positive for M. plutonius using real-time PCR. A two-tailed T-test on paired quantitative data suggested no difference in the relative amount of M. plutonius between foraging and hive bees (p=0.81).

Dissections of adult bees revealed all external and alimentary organs were positive for M. plutonius. Quantitative analysis demonstrated levels of M. plutonius were substantially higher in the rectum, compared to other body parts tested: the rectum contained 20 times more bacteria than the head, and 7 times more than the crop and ventricles. This difference was significant (P<0.05), even when analyses were adjusted to allow for multiple comparisons.

Samples of larvae (n=61) and adult bees (n=65) were collected from the nearest known apiaries to a new outbreak in Cumbria. All samples of larvae tested negative for M. plutonius using real-time PCR, however two adult bee samples tested positive using real-time PCR (Average CTs 37.80 & 38.11). Additional DNA extractions prepared from remaining adult bee samples again tested positive, demonstrating these results were not false positives. Both positive samples were from the same apiary that was within 3 km of the initial outbreak site. Follow up inspections in 2009 demonstrated the absence of EFB symptoms, suggesting the disease did not establish in Cumbria.

Dissemination of results (O6)The results from this project formed a key part of the NBU presentations to industry and scientists from 2007. These are listed in Section 9 of this report. In addition, an A4 summary of the research findings was completed and handed directly to beekeepers by inspectors, and also added to the Research and Development pages of BeeBase. Bee inspectors also presented results at regional training events since spring 2007. A manuscript summarising the main findings has been submitted to a peer-reviewed journal for consideration.

Trends for treatment the selection for EFB control method by beekeepers were monitored in England and Wales. The results showed a clear positive shift from OTC to shook swarm treatments over time (Figure 7).

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Figure 7. Trend for the selection of EFB treatments by beekeepers. Extension of project results began in spring 2007

Overall DiscussionThis project has provided evidence to suggest that SS presents a viable alternative to OTC for the control EFB in England and Wales. In the spring following treatment shaken colonies were significantly less likely to test positive for M. plutonius (Figure 1B). In addition shaken colonies were less likely to have symptoms of EFB than OTC treated colonies in the year following treatment. Data directly comparing shook swarm and OTC treatments have not been presented previously. As an antibiotic, OTC requires a withdrawal period of at least 16 weeks to avoid excessive residues in honey (Thompson et al., 2006). Honey flows coinciding with withdrawal periods can lead to a large loss in honey productivity. The shook swarm method presented here has the advantage that no antibiotics are used, negating the risk of antibiotic contamination of honey after treatment. There are several hundred confirmed cases of EFB in England and Wales annually that require considerable resources to fund apiary inspections, diagnoses and management (Wilkins et al., 2007). EFB is endemic in Switzerland, and cases of the disease have increased 10-fold over the last 10 years (Roetschi et al., 2008). Sanitisation procedures in Switzerland involve destruction of every symptomatic and weak colony, autoclaving of combs and the disinfection of beekeeping materials with 5% sodium carbonate or 4% sodium hydroxide. However, this sanitation protocol was found to be inadequate for eliminating M. plutonious from EFB affected apiaries (Roetschi et al., 2008). Adopting a control method which reduces disease reoccurrence will save considerable resources treating EFB outbreaks in the UK, and also may be applicable in other countries like Switzerland. Both applications would lead to healthier bee stocks and help to protect the vital role of honey bees as crop pollinators.

Quantitative real-time PCR was used to demonstrate bacterial levels prior to treatment differed significantly between symptomatic and asymptomatic colonies (Figures 2 and 4). Such data suggest disease thresholds may occur with EFB infection, corroborating recent data from Switzerland (Roetschi et al., 2008). Our qualitative data clearly demonstrate that there is an increased risk of M. plutonius being present in asymptomatic colonies from apiaries with EFB present compared to asymptomatic colonies in apiaries free from EFB (Figure 1A), a finding consistent with an earlier report (Belloy et al., 2007). A field trial was conducted in 2007 to observe differences between apiaries where SS was applied to only symptomatic colonies and EFB-affected apiaries where all colonies were treated with SS. Due to poor weather, the trial was half the planned size. Despite this, the whole apiary SS clearly demonstrated a significant reduction in the number of colonies testing positive for M.

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plutonius, post treatment (Figure 5), and there was a lower frequency of disease reoccurrence, although this did not reach statistical significance (Figure 6A). This finding suggests the detectable inoculum on adult bees from contact colonies can be successfully manipulated by changing the brood comb.

Shaking colonies is an old idea for disease control that was widely adopted prior to the advent of antibiotics. The SS treatment emulates the natural behaviour of the honey bee to move nest site when disease pressure is high, thereby reducing the build-up of disease agents. Comb replacement into a clean box presents a disease free environment, and forces a break in the brood cycle that also provides a break in the disease cycle. Shaking is known to work for other bacterial diseases such as American foulbrood, where reinfection rates of about 5% have been recorded (Shimanuki & Knox, 1997). Ensuring shaken colonies have access to ample nutrition by supplemental feeding and proper treatment timing are both essential for colony survival. Guler (2008) reported that shaking colonies 45 days before the main nectar flow with no supplementary feeding reduced honey yield and led to high colony mortality. Such data demonstrate the importance of supplemental feeding and applying shook swarm treatment at a time of year when bees are able to draw comb and store sufficient honey for winter periods (Shimanuki & Knox, 1997).

Historically EFB prevalence is low in the north of England and Wales compared to the south. Two hypotheses would support such an observation. Firstly, the low prevalence in the north of England and Wales could be due to the absence of the causal agent of EFB, M. plutonius. Secondly, M., plutonius could be ubiquitous across England and Wales but the environmental conditions in the north are not conducive for disease development. Belloy et al. (2007) presented data suggesting M. plutonius was absent from two apiaries located in an area of Switzerland free from EFB, suggesting M. plutonius is not ubiquitous in Switzerland. In the current study, all 18 EFB free apiaries located in North England and North Wales, where EFB does not occur, tested negative for M. plutonius in 2006. In 2007, 62 of 64 samples from apiaries in Cumbria also tested negative for M. plutonius. These data provide evidence that M. plutonius may not be ubiquitous in England and Wales. Interestingly, one apiary in Northern England tested weakly positive for the presence of M. plutonius. The apiary was situated within 3 km of the only confirmed EFB-affected apiary in the county. The M. plutonius positive samples were collected two weeks after the arrival of the EFB-infected colonies into the region, demonstrating how rapidly the bacterium can distribute to neighbouring apiaries. These data suggest either long-distance adult bee transfer, perhaps during robbing, or the potential of contamination of shared forage.

In the current project we found that flying bees contained similar levels of inoculum to hive bees. Whist this experiment was small, the results are in stark contrast to reports from Switzerland where hive bees from EFB-affected colonies contained 20 times more inoculum than flying bees (Roetschi et al. 2008). Such data suggests the inoculum frequently detected in adult bees could be more significant to disease spread than previously thought. Real-time PCR could be an effective method of screening forage plants around infected apiaries to investigate whether M. plutonius is present on and around inflorescences.

Dissections revealed the distribution of inoculum in adult bees, suggesting all external and alimentary organs were positive for M. plutonius. Our results confirm previous findings in adult bees from EFB colonies using hemi-nested PCR (McKee et al., 2003). In addition, the honey bee rectum has been reported to contain more anaerobic microorganisms than the midgut (Kačániová et al., 2003). However, this is the first report to use a quantitative approach to demonstrate the bacterium is more common in the rectum of the adult honey bee. These data support current thinking that EFB has a faecal-oral route of transmission (Bailey, 1959). The epithelium of the honey bee’s rectum is highly folded, allowing the rectal sac to distend to hold an immense accumulation of faecal matter and excretory products from the associated Malphigian tubules (Snodgrass, 1956; Dade, 1977). Bees never normally defecate within the hive, and during long cold periods (i.e. in the winter, when they are unable to leave the colony) they are able to retain so much intestinal waste that the rectum becomes expanded to an enormous bag occupying all available space in the abdomen (Dade, 1977; Graham, 1992). In healthy honey bees, faeces largely consist of pollen husks, pollen fat globules, and the exhausted epithelial cells of the ventriculus (stomach). However, when bees are diseased, fed an inappropriate diet, or are confined to their hives for extreme lengths of time, these substances will ferment in the rectum, and bacteria, yeasts and microfungi will thrive in enormous numbers (Dade, 1977). The resulting irritation causes dysentery, and potentially infected waste matter is involuntarily expelled inside the hive (Dade, 1977). Dysentery leaves tell-tale “spotting” in and around affected colonies. Although spotting is not a characteristic symptom in EFB-infected hives, it was observed that each one of the EFB-positive bees dissected for this project had a very full rectum. This suggests that EFB infection may contribute to accumulation of faecal matter in the posterior reaches of the host bee’s digestive tracts, which then act as reservoirs for infective M. plutonius. Further study is required to confirm these observations

The data presented in this study provides useful information comparing options for EFB control. The long-term monitoring of disease reoccurrence remains the best guideline for the assessment of treatment efficacy for the control of EFB. This project report also presents interesting data to supplement our poor understanding of EFB epidemiology. Such data are complemented by findings in related project PH0505 investigating the role(s) of secondary bacteria. The DNA collections from this project remain stored and could provide useful insight into the role played by other bacteria, like P. larvae, to further inform about this damaging brood disease of the honey bee. The data generated in this project will assist with improved, evidence-based management of EFB in England and Wales.

Policy relevance

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The data generated within this project clearly demonstrate husbandry methods offer a suitable alternative to antibiotic usage for the control of EFB. As such, this project has changed the advice provided to beekeepers by the National Bee Unit. The NBU now advises that, where possible, SS should be employed in place of OTC. OTC usage still has a place for the containment of EFB whilst awaiting suitable environmental conditions to apply the SS treatment. However, this work was timely because OTC usage for the control of EFB could be withdrawn in the future. SS adopts a cost sharing partnership with the beekeeping industry whereby Defra funds apiary inspections, treatment application and monitoring and beekeepers cover the cost of replacement equipment (mainly frames and foundation). Beekeepers also have the option to cover the cost of comb replacement by taking out insurance schemes which cover the costs incurred by treating for EFB (cost from £2 per annum to insure three colonies). This project produces some data which may indicate that the widespread adoption of SS (over OTC) could reduce the long term incidence of EFB, and therefore reduce the cost to Defra for managing this disease, and allowing bee inspectors to spend more time training and educating beekeepers. However, this needs to be confirmed by monitoring treatment efficacy data at the apiary level using BeeBase.

References

Aleksandrova, L.V. (1949) Works of the Veterinary Section of the Lenin Academy of Agricultural Science Session 37: 148.

Bailey, L. (1960) J. Insect Pathol. 1: 80-85.Bailey, L. (1960) J. Insect Pathol. 2: 67-83.Bailey, L. (1961) American Bee Journal 101: 89-92.Bailey L. (1983) J. Appl. Bacteriol. 55: 65-69.Bailey L., Locher N. (1968) J. Apic. Res. 7: 103-107. Bailey, L., Collins, M.D. (1982) J. App. Bacteriol. 53: 215-217.Belloy, L., Imdorf, A., Fries, I., Forsgren, E., Berthoud, H., Kuhn, R., Charriere, J.D. (2007) Apidologie 38: 136-

140.Borneck, R., Merle, B. (1989) Apiacta 24: 33-38.Carreck, N., Williams, I. (1998) Bee World 79: 115-123.Dade (1977) Anatomy and Dissection of the Honey Bee. IBRA, London.Gil-Salas, F.M., Morris, J., Colyer, A., Budge, G., Boonham, N., Cuadrado, I.M., Janssen, D. (2007) J. Virological

Methods 146: 45-51.Gordon, J., Davis, L. (2003) Rural Industries Res. & Devel. Corporation Australia RIRDC Report 03/077. 1983-

1985.Graham, J.M, (1992) The hive and the Honey bee. Dadant & Sons. Illinois USA. pp1324.Guler, A. (2008) J. Apic. Res. 47: 27-34Office International des Epizooties (2006) Annual disease status pages.

http://www.oie.int/eng/info/en_infoan.htm. Verified 13/02/2006.Kacániová, M., Chlebo, R., Kopernický, M., Trakovická, A. (2004). Microflora of the honeybee gastrointestinal

tract. Folia microbiologica 49: 169-71.Mckee, B.A., Djordjevic, S.P., Goodman, R.D., Hornitzky, M.A. (2003) Apidologie 34: 19-27.Matheson, A. (1993) Bee World 74: 176–212. Morse, R. A., Calderone, N.W. (2000) Bee Culture 128: Supp. 1-15.Roetschi, A., Berthoud, H., Kuhn, R., Imdorf, A. (2008) Apidologie 39: 362-371.Shimanuki H. (1997) In: Morse R.A. and Flottum K. (Eds.), Honey Bee Pests, Predators, and Diseases, The A.I.

Root Company, Medina, Ohio, USA. Shimanuki, H., Knox, D.A. (1997) In: Morse R.A. and Flottum K. (Eds.), Honey Bee Pests, Predators, and

Diseases, The A.I. Root Company, Medina, Ohio, USA. Snodgrass, R.E. (1956) Anatomy of the Honey Bee. Constable & Co. Ltd., London.Thompson, H., Brown, M.A. (2001) Bee World 82: 130-138.Thompson, H.M., Waite, R.J., Wilkins, S., Brown, M.A., Bigwood, T., Shaw, M. et al. (2006) Apidologie 37: 51-57.Waite, R.J., Brown, M.A., Thompson, H.M., Bew, M.H. (2003) Apidologie 34: 569-575.Ward, L., Waite, R., Boonham, N., Fisher, T., Pescod, K., Thompson, H. et al. (2007) Apidologie 38: 181-190.Ward, L., Brown, M., Neumann, P., Wilkins, S., Pettis, J., Boonham, N. (2007) Apidologie 38: 272-280.Wilkins, S., Brown, M.A., Cuthbertson, A.G.S. (2007) Pest Management Science 63: 1062-1068.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

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List of key presentations where project results formed a major proportion of the content(MB – Mike Brown, GB – Giles Budge)

Date Presenter Platform/group Approx. no. of attendees

03/07 GB Bee inspectors conference 8003/07 MB West Sussex Beekeeping Convention 10003/07 MB Cambridgeshire Beekeepers Conference 10003/07 MB Northern Branch BFA York 10004/07 MB Bee Inspectors Conference York 7004/07 MB AFSSA Paris 3011/07 GB Central association of beekeepers 7003/08 GB BFA annual convention 7003/08 GB BIBBA spring convention 20004/08 GB & MB Bee inspectors conference York 8004/08 GB & MB Stoneleigh (Poster/Stand) 2,00005/08 GB Eastern region bee forum 2008/08 GB & MB OIE, Freiburg 25009/08 GB & MB Coloss 4009/08 GB & MB Café Scientific 10009/08 GB & MB New Agency Inspectorate 10010/08 GB EurBee Conference, Belfast 30010/08 GB National Honey Show 20010/08 GB Bee Farmers Association 20011/08 MB WAG meeting Builth Wells 4011/08 MB DARG meeting Buckfastleigh Devon 4012/08 MB Whitby BKA 2012/08 GB Annual Beekeepers Meeting 10003/09 MB Various presentations 35004/09 GB & MB Bee inspectors conference York 10005/09 MB Eastern Region Forum 4006/09 MB AFSSA Paris 100

In addition to above presentations, an A4 summary of the research findings was completed and handed directly to beekeepers by inspectors, and also added to the Research and Development pages of BeeBase.

Bee inspectors also presented results at regional training events since spring 2007.

A manuscript summarising the main findings has been submitted to a peer-reviewed journal for consideration.

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Documents

General enquiries on this form should be made to:sciencesearch.defra.gov.uk/Document.aspx?Document=PH0502... · Web viewAs with other forms of livestock, honey bees are subject to