Transmission of American Foulbrood (AFB) Disease …...escorts, 2 (13.3%) survived and 13 (86.7%)...

Transmission of American Foulbrood (AFB) Disease of Honeybees Through Replacement of Queen bees

A report for the Rural Industries Research and Development Corporation

by Patricia Greer January 2007 RIRDC Publication No 07/028 RIRDC Project No DAQ-293A

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© 2007 Rural Industries Research and Development Corporation. All rights reserved. ISBN 0 642 1 74151 428 2 ISSN 1440-6845

Transmission of American foulbrood (AFB) disease of honeybees through replacement of queen bees Publication No. 07/028 Project No. DAQ-293A The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186. Researcher Contact Details Patricia Greer Department of Primary Industries and Fisheries Gatton Research Station Locked Mail Bag 7 MS 437 Gatton Qld 4343 Phone: 07 5466 2216 Fax: 07 5462 3223 Email: [email protected]

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6272 4819 Fax: 02 6272 5877 Email: [email protected]. Website: http://www.rirdc.gov.au Published in January 2007 Printed on environmentally friendly paper by Canprint

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Foreword American Foulbrood (AFB) is a serious bacterial disease of honeybees in Australia, causing major economic loss to apiarists. Infection is known to occur in a variety of ways including ingestion of contaminated honey or pollen, the interchange of contaminated equipment between colonies and drifting of bees between colonies. The aim of this study was to determine, under field conditions, whether clinical AFB disease could result if queen bees were transferred from infected to clean colonies. Queen bees are a main expense for commercial and amateur apiarists, and are usually replaced annually. Anecdotal evidence from apiarists suggests that repeated AFB outbreaks is associated with queen bee replacement. This is contrary to advice by leading queen bee providers. Some studies have suggested queen bees are capable of disseminating enough spores from their alimentary canals to play a role in causing epizootic cases among the larvae of honeybees. Other studies suggest queens are a low risk in the spread of AFB. However, no field trials have been conducted to prove or disprove these theories. The key outcomes of this project demonstrate that queen bees, taken from infected hives and used to requeen uninfected colonies, are unlikely to cause clinical AFB in those colonies, and hence, this report will be useful to Australian apiarists in determining whether to use queen bees from infected hives in new hives. This project was funded from industry revenue which is matched by funds provided by the Australian Government. This project was funded from industry revenue which is matched by funds provided by the Australian Government. This report, an addition to RIRDC’s diverse range of over 1500 research publications, forms part of our Honeybee R&D program, which aims to improve the productivity and profitability of the Australian beekeeping industry. Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/fullreports/index.html • purchases at www.rirdc.gov.au/eshop Peter O’Brien Managing Director Rural Industries Research and Development Corporation

http://www.rirdc.gov.au/fullreports/index.html

http://www.rirdc.gov.au/eshop

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Acknowledgments We thank Ross Newman, Senior Veterinary Officer for his assistance with all aspects of the research. We thank Wendy Ward and Paul Burrell, bacteriologists, for their excellent laboratory work. We thank Department of Primary Industries and Fisheries, Queensland & Forestry for use of a forestry area site. We also thank the beekeepers who assisted with hives and queen bees.

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Contents Foreword ................................................................................................................................................ iii Acknowledgments .................................................................................................................................. iv Executive Summary ............................................................................................................................... vi 1. Introduction ........................................................................................................................................ 1 2. Objective ............................................................................................................................................ 2 3. Materials and Methods ....................................................................................................................... 3

3.1 Apiary........................................................................................................................................... 3 3.2 Treatment Protocols ..................................................................................................................... 3 3.3 Preliminary Examination of Hives ............................................................................................... 3 3.4 Infection with American foulbrood .............................................................................................. 4 3.5 Transfer of the queen bees............................................................................................................ 4 3.6 Culture of Paenibacillus larvae subsp larvae .............................................................................. 4 3.7 Examination of the hives.............................................................................................................. 5

4. Results ................................................................................................................................................ 6 4.1 Treatment 1 – Queens only without escorts ................................................................................. 6 4.2 Treatment 2 – Queens with escorts .............................................................................................. 6

5. Discussion .......................................................................................................................................... 7 References ............................................................................................................................................... 8 Appendices .............................................................................................................................................. 9

Appendix 1 .......................................................................................................................................... 9 Appendix 2. ....................................................................................................................................... 11

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Executive Summary Background American Foulbrood (AFB) is a serious bacterial disease of honeybees in Australia, causing major economic loss to apiarists. Infection is known to occur in a variety of ways including ingestion of contaminated honey or pollen, the interchange of contaminated equipment between colonies and drifting of bees between colonies. Queen bees are a main expense for commercial and amateur apiarists, and are usually replaced annually. Anecdotal evidence from apiarists suggested repeated AFB outbreaks were associated with queen bee replacement. This was contrary to advice by leading queen bee providers. Some studies have suggested queen bees were capable of disseminating enough spores from their alimentary canals to play a role in causing epizootic cases among the larvae of honeybees, other studies have suggested queens may be a low risk in the spread of AFB. However, no field trials have been conducted to prove or disprove these theories. Objectives The aim of this study was to determine, under field conditions, whether clinical AFB disease could result if queen bees were transferred from infected to clean colonies. Methods Ninety six nucleus colonies in two apiaries under field conditions were used to determine whether the transfer of queen bees from American foulbrood affected hives to non infected hives would result in transmission of the clinical disease. Two treatments were used, with neither treatment resulting in clinical infection of colonies. Results The first treatment involved requeening using queens only. Of the 17 hives requeened without escorts, 3 (17.6%) survived and 14 (82.4%) died out. The adult bees from one hive carried Paenibacillus larvae (AFB) spores at the first sampling after requeening. On the second and third sampling, results could not be obtained for 1 and 5 hives respectively. None developed clinical AFB symptoms. The second treatment involved requeening using queens and escorts. Of the 15 hives requeened with escorts, 2 (13.3%) survived and 13 (86.7%) died out. The adult bees from three different hives carried Paenibacillus larvae subsp larvae (AFB) spores at various samplings after requeening. On the second and third sampling, results could not be obtained for 4 and 5 hives respectively. None developed clinical AFB symptoms. (The dead hives had either failed to requeen, the queens had failed or the hives had starved. Where no result could be obtained, this was due to contamination during culturing). The adult bees from one control hive also carried Paenibacillus larvae spores at the first sampling after requeening. This hive had failed to requeen, and died. Of the five hives showing P.l. larvae spores on adult bees, three of these occurred in the first sampling after requeening, one in the second sampling and one in the third sampling. Samplings were approximately one month apart, with the first sampling one month after requeening. Six samplings were conducted, with the trial being discontinued with 12 hives remaining. The trial was discontinued at six months due to drought conditions and shortage of food, lack of suitable alternative sites, and the poor condition of the remaining colonies.

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Implications This study demonstrated that queen bees, taken from infected hives and used to requeen uninfected colonies, are unlikely to cause clinical AFB in those colonies. However, it is not certain whether more favourable conditions together with a longer timeframe may have produced an alternative outcome. This uncertainty, coupled with the fact that 27 (84.4%) of the 32 treatment hives died in the trial indicates further experimental work would be advisable to confirm these findings.

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1. Introduction American Foulbrood (AFB) is a serious bacterial disease of honeybees in Australia, causing major economic loss to apiarists. Infection is known to occur in a variety of ways including ingestion of contaminated honey or pollen, the interchange of contaminated equipment between colonies and drifting of bees between colonies. Queen bees are a main expense for commercial and amateur apiarists, and are usually replaced annually. Anecdotal evidence from apiarists suggested repeated AFB outbreaks associated with queen bee replacement. This was contrary to advice by leading queen bee providers. This project will determine whether queens from infected hives are capable of transmitting AFB to disease free hives under Australian field conditions. Several studies have suggested queen bees may be a low risk in the spread of AFB (Hornitzky 1991; Goodwin, et al 1993). However, no trials have been conducted to prove or disprove this theory. Al Zubaidy and Wilson 1980 found queens were capable of excreting and disseminating enough spores from their alimentary canals to play a role in causing epizootic cases among the larvae of honeybees. However, no field trials have been conducted to prove or disprove these theories.

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2. Objective To better understand the role queen bees’ play in transmitting AFB to managed honeybee colonies.

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3. Materials and Methods 3.1 Apiary Initially sixty two nucleus colonies were established at the Animal Research Institute (ARI) in Yeerongpilly, Brisbane. Requeening problems reduced this to 48 hives. Sixteen of these were controls, with the balance treatments. Each hive was requeened with a young, vigorous queen purchased from a commercial queen bee breeder. The colonies were on good food sources, with many colonies displaying two - four frames of brood each, and top bars completely covered with bees upon opening the colony. A further sixty nucleus colonies were established at Esk State Forestry, Esk, Queensland. Before Esk was chosen as a suitable site, test hives were placed in position near the centre of the forestry and tested for AFB for 6 months. No other apiaries were sited in the forestry. No AFB was detected in the 6 month period. These hives were on good food sources initially, before deteriorating into very poor conditions.

Photo 1: Nucleus colony at Esk Forestry

Photo 2: Nucleus colonies established at Esk Forestry.

3.2 Treatment Protocols Two treatments were employed: 1. Queen bees with escorts. 2. Queen bees without escorts. 3.3 Preliminary Examination of Hives All hives at ARI and at Esk were examined for clinical symptoms and adult bees sampled and cultured for AFB prior to starting the trial. These were found to be clear of AFB.

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3.4 Infection with American foulbrood To achieve AFB infection, 8002 cm of young brood per hive were inoculated with a total of 10 mls of suspension containing 4.8 x 109 P. larvae spores. (Hornitzky 1998). This was repeated after 5 weeks to achieve uniform infection. The strength of the hives and levels of infection achieved are detailed in Appendix 2.

Photo 3: Spraying young brood with

American foulbrood innoculum 3.5 Transfer of the queen bees Once 100% infection was achieved, in the hives at ARI, the apiary from Esk was returned to ARI for 3 weeks. All queens in the Esk hives were killed on one day, and the queens from ARI introduced the following day. Queens from control hives were caught, caged and introduced first and then the treatments were caught, caged, and introduced. Even numbered hives received queens and escorts, and odd numbered hives were queens only. All queens were marked with paint prior to caging. 3.6 Culture of Paenibacillus larvae subsp larvae Adult bees were cultured as described by Hornitzky (1998).

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3.7 Examination of the hives The first examination occurred 4 weeks after queen introduction. The hives were removed to Esk Forestry, and sampled every month were practicable. The results of each sampling are detailed in Appendix 1.

Photo 4: Sample of 30 bees taken or American foulbrood analysis.

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4. Results Forty eight queens were transferred into clean hives with a loss of 13 queens. Of these, 8 hives reared queens and were continued in the trial. The 5 queenless hives were removed. 4.1 Treatment 1 – Queens only without escorts

• 17 hives were requeened • 3 (17.6%) survived for six months • 14 (82.4%) died out • 1st sampling – 1 hive positive for P.l.larvae spores • 2nd sampling no result could be obtained for 1 sample • 3rd sampling, no result could be obtained 5 samples • No hive developed clinical AFB symptoms.

4.2 Treatment 2 – Queens with escorts

• 15 hives were requeened • 2 (13.3%) survived for six months • 13 (86.7%) died out • 1st sampling – 1 hive positive for P.l.larvae spores • 2nd sampling – 1hive positive for P.l.larvae spores

- no result could be obtained for 4 samples • 3rd sampling - 1 hive positive for P.l.larvae spores

- no result could be obtained 3 samples • No hive tested positive more than once • No hive developed clinical AFB symptoms.

(The dead hives had either failed to requeen, the queens had failed or the hives had starved. Where no result could be obtained, this was due to microbial contamination during culturing). Table 1: Samplings which tested Positive for P.l. larvae, or where no result could be obtained.

Treatment 28 April 05 31-May-05 14-Jul-05 10-Aug-05 05-Sep-05 13-Oct-05 +ve* +ve +ve +ve +ve +ve

1. Treatment with escorts

1+ve 1+ve (4) 1+ve (3)

2. Treatment without escorts

1+ve (1) (5)

3. Control with escorts

(1) (4) (5) (2)

4. Control without escorts

1+ve (1) (1)

* Positive (x) No. where no result could be obtained The adult bees from one control hive also carried Paenibacillus larvae spores at the first sampling after requeening. This hive had failed to requeen, and died. Samplings were approximately one month apart, with the first sampling one month after requeening. Six samplings were conducted, with the trial being discontinued after six months with 12 hives remaining. This was due to extreme dearth conditions, lack of suitable alternative sites, and the poor condition of the remaining colonies.

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5. Discussion The aim of this study was to determine, whether clinical AFB disease could result if queen bees were transferred from infected to clean colonies. Two protocols were used under field conditions in an AFB free area at Esk in Queensland. None of the hives developed clinical AFB. The first protocol involved requeening hives using queens without escorts, while the second involved using queens with escorts. Of the five hives showing P.l. larvae spores on adult bees, three of these occurred in the first sampling after requeening, one in the second sampling and one in the third sampling. Spores persisted in the group with escorts, although no hive tested positive more than once. This result shows that potentially, significantly more spores are transferred if escorts are used, and these spores can persist for some time. Samplings were approximately one month apart, with the first sampling one month after requeening. Six samplings were conducted, with the trial being discontinued with 12 hives remaining. This study demonstrated that queen bees, taken from infected hives and used to requeen uninfected colonies, are unlikely to cause clinical AFB in those colonies. However, it is not certain whether more favourable conditions together with a longer timeframe may have produced an alternative outcome. This uncertainty, coupled with the fact that 27 (84.4%) of the 32 treatment hives died in the trial indicates further experimental work would be advisable to confirm these findings.

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References Al Subsidy M. and Wilson W.T. (1980). Persistence of Bacillus larvae W., in alimentary canal of

queen honey bees, Apis mellifera. Bull Nat Hist Res Centre 7(4): 1–10. Goodwin R.M., Perry J.H. and Brown P. (1993). American foulbrood disease, Part III: Spread. The

New Zealand Beekeeper 219: 228–235. Hornitzky M. (1998). The spread of Paenibacillus larvae subsp larvae infections in an apiary.

Journal of Apicultural Research 37(4): 261–265. Hornitzky M. (1991). Can American brood disease be transmitted by a queen bee and her escorts? The

Australasian Beekeeper January pp 271.

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Appendices Appendix 1 This appendix provides data on all of the hives used in the experiments after inoculation and transfer of queens into disease free hives. Hive 28 April 05 31-May-05 14-Jul-05 10-Aug-05 05-Sep-05 13-Oct-05 Number Results Results Results Results Results Results

1 Self Raised Queen neg neg no result neg neg dead

3 Queenless neg

4 Q Okay * neg neg no result neg neg dead

5 Self Raised Queen neg Drone Layer

7 Q Okay not marked? neg neg no result neg neg dead

8 Q Okay not marked? neg neg no result neg neg dead

9 Self Raised Queen 1+ neg no result neg neg dead

11 Q Okay neg neg no result neg dead neg

12 Q Okay neg no result neg neg neg neg

13 Q Okay neg neg neg neg neg dead

15 Q Okay neg neg no result neg dead dead



19 Q Okay neg neg neg neg neg neg

20 Q Okay neg no result no result neg neg dead

21 Queenless neg

22 Self Raised Q neg Drone Layer

24 Q Okay neg neg neg dead dead dead

25 Q not marked? neg neg neg neg neg dead

26 Q Okay neg 2+ (26 colonies) no result neg neg dead

27 Self Raised Queen neg Dead dead

28 Q Okay neg no result no result neg neg neg


30 Queen Less neg


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Hive 28 April 05 31-May-05 14-Jul-05 10-Aug-05 05-Sep-05 13-Oct-05 Number Results Results Results Results Results Results

32 Q Okay neg neg no result neg neg dead

33 Self Raised Queen neg neg neg neg neg dead

34 Q Okay 1+ neg neg neg neg dead

35 Q Okay neg no result no result neg dead dead

36 Q Okay neg no result neg no result neg neg



40 Q Okay neg no result no result neg neg neg

41 Q Okay neg no result neg neg neg neg

43 Queenless 1+




47 Q Okay neg neg no result neg neg neg

49 Queenless neg

50 Q Okay no result neg neg neg dead dead


52 Q Okay neg no result neg no result neg neg


54 Q Okay neg no result neg neg neg dead


57 Q Okay neg neg neg neg dead dead

62 Q Okay neg no result 1+ dead dead dead • Grey Shaded cells are controls • Even numbers received escorts • Odd numbers were queens only without escorts • *Queen successfully introduced • 1+ indicated one colony of P.l.larvae was cultured for that hive. • 2+26 indicated 26 colonies of P.l.larvae were cultured for that hive.

Column 1 indicates hive number Column 2 indicates health of the queen and/or hive at first inspection after introduction. Remaining columns give results of samplings.

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Appendix 2. This appendix provides data on the hives which were inoculated with P.l.larvae, indicating strength and level of infection of the hives from which the queens initiated.

Hive No. Q's 23/09/04 Q's 08/10/04 Q’s21/10/04 Q's 05/11/04 Q's 29/11/04 Q's 10/03/05 31/03/05 2005

Treated No. diseased cells in egg ring Pop Rate Brood Count AFB Level

1 Q Q 4.8x109 14, 6, 19, 24 W'out escorts M 3, 3, 3, 2 L 2 Q Q 4.8x109 Qless

3 Q Control W'out escorts 4 Q Control With Escorts

5 Q Q Q Q 4.8x109 5, 0, 6, 1 W'out escorts M 3, 2, 2, 2 M 6 Q Q 4.8x109 Qless

7 Q 4.8x109 0, 2, 4, 0 W'out escorts H 3 3 2 2 M 8 Q 4.8x109 1, 9, 6, 3 With Escorts M 3 3 0 3 L

9 Q 4.8x109 4 3 5 3 W'out escorts L 2 0 2 2 L

10 Q 4.8x109 2 0 12 14 With Escorts M 2 2 0 1 L


13 Q 4.8x109 1 3 4 0 W'out escorts H 3 3 2 3 L 14 Q 4.8x109 Q Cell 7 9 0 0 Qless

15 Q Q 4.8x109 0 3 3 4 W'out escorts L 1 3 3 1 M 16 Q Q 4.8x109 1 0 1 0 With Escorts H 2 2 0 2 L

17 Q dead 18 Q 4.8x109 0 0 0 15 With Escorts H 1 2 3 1 L


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21 Q 4.8x109 1 3 4 3 W'out escorts M 1 3 3 3 L 22 Q 4.8x109 0 0 9 1 With Escorts H 3 3 1 2 L

23 Q 4.8x109 Dead 24 Q 4.8x109 0 0 0 0 With Escorts H 2 3 3 2 L

25 Q 4.8x109 0 2 1 8 W'out escorts H 0 3 3 2 L 26 Q 4.8x109 5 0 1 0 With Escorts H 2 2 2 0 M


29 Q Self R Q Q less? 8 7 2 3 W'out escorts H 3 2 3 3 L 30 Q 4.8x109 8 3 2 2 With Escorts M 3 3 0 3 M

31 Q Self R Q Control W'out escorts 32 Q Self R Q Control With Escorts

33 Q 4.8x109 0 0 6 13 W'out escorts H 3 2 3 2 M 34 Q 4.8x109 4 0 3 3 With Escorts M 3 2 3 3 M


37 Q Dead 38 Q 4.8x109 1 5 2 3 With Escorts H 3 3 3 2 M

39 Q 4.8x109 3 6 7 7 W'out escorts M 3 3 3 3 H 40 Q 4.8x109 2 0 3 0 With Escorts H 3 3 2 2 L

41 Q 4.8x109 0 0 1 5 W'out escorts H 3 3 1 2 L 42 Q Dead


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45 Q 4.8x109 2 2 2 0 W'out escorts H 2 2 3 3 L 46 Q 4.8x109 4 3 8 0 With Escorts H 2 3 3 1 L

47 Q 4.8x109 0 3 0 1 W'out escorts H 2 3 3 2 L 48 Q 4.8x109 0 0 1 2 With Escorts H 2 3 2 1 L

49 Q 4.8x109 1 0 0 0 W'out escorts M 0 2 2 1 L 50 Q 4.8x109 1 15 26 7 With Escorts M 1 2 2 0 M

51 Q Control 52 Q Control

53 Q 4.8x109 3 1 8 15 W'out escorts M 3 3 3 0 H 54 4.8x109 0 1 1 0 With Escorts H 3 3 3 1 H

55 Q 4.8x109 dead 56 Q 4.8x109 1 0 13 5 M 2 3 3 2 H


59 Q 4.8x109 Dead 60 Q 4.8x109 Dead

61 Q 4.8x109 3 0 1 2 W'out escorts L 3 1 0 0 M 62 Q 4.8x109 2 4 3 0 With Escorts M 3 3 3 3 M

• Column 1 indicates hive number • Column’s 2-5 indicate when queens (denoted by Q), purchased from a commercial breeder were introduced • Column 6 indicates hives treated with inoculum • Column 7 indicates the number of diseased brood cells in a randomly placed egg ring place over a brood patch in the hive • Column 9 indicates population - Light (2 frames or less) Medium - (Good but not over topbars) Heavy (bubbling over topbars) • Column 10 is the amount of brood present - 3 = three quarters or more of frame, 2= half to three quarters of frame, 1 = up to half of a frame • Column 11 indicates level of AFB infection - Light - <one quarter of all brood infected, Medium - one quarter to one half of all brood infected, Heavy - > one half of all brood

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Transmission of American Foulbrood (AFB) Disease …...escorts, 2 (13.3%) survived and 13 (86.7%)...

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Transcript of Transmission of American Foulbrood (AFB) Disease …...escorts, 2 (13.3%) survived and 13 (86.7%)...