• To allow all industry participants, from the producer through to the packer, including relevant state government departments, to work in a unified manner developing strategic plans to manage the reduction in the level of AFB and EFB in Australia’s honeybee population. 
• Coupled with the strategic plan are guidelines for the use of chemicals to ensure that their usage does not present an unacceptable residue level in Australian honey.

Unfortunately, industry now finds itself with unacceptable levels of AFB and the risk of chemical residues.

Drivers were challenged to report to the various state and sector annual conferences.

• To identify critical areas during the production of queen bees which influence introduction and early performance levels by sampling and recording conditions under which queen bees are reared during spring 1997 and autumn 1998. 
• Data from this project and from the project DAN-164A "Introduction of queen bees - introductory apiary status and post introduction results" to be analysed together.

Only members of the AQBBA agreed to participate in the survey.

Nutrition, disease and population strength were recorded and samples taken for examination.

All apiaries and stages were considered to have adequate nutritional stores.

All apiaries and stages were considered to have adequate population for the purpose required.

All mating yards examined were considered to have adequate numbers of drone mother hives in close proximity. The drone mother hives were regarded to have the quantity of drones required.

No brood diseases were reported.

The survey of queen bee breeder apiaries did not reveal any obvious areas in the production of queen bees that would influence the quality of queen bees produced.

Nosema disease was found in one queen out of 59 examined.

Some sperm counts were lower than expected even though drone mother hives when examined were considered to have adequate numbers of drones present. (spring survey)

• To isolate and identify the ingredients of the natural attractant that the Asian hive bee releases as a homing signal (aggregation pheromone) for other members of its species; 
• To make a synthetic mixture that mimics this pheromone; and 
• To show that the blend can specifically attract this pest.

• To determine the oxytetracycline hydrochloride sensitivity of Australian Melissococcus pluton isolates.
• To determine the diversity of Australian M. pluton isolates.
• To determine whether M. pluton isolates contain plasmids (antibiotic resistance transfer vectors).

In studies carried out in the early 1960s strains of M. pluton were determined to be closely related to each other in spite of their widely separate geographical origins. In recent years molecular typing technologies have advanced the capability of differentiating bacterial, viral and parasite isolates within the same species or genus. The DNA analysis of M. pluton isolates will be useful in determining whether the isolates from areas where EFB appears to be more severe than in other areas have different DNA profiles. If severity of disease can be associated to a particular DNA profile it may be possible to predict increases in disease severity if more virulent strains are detected in areas where they had previously not been recognised. It would also be useful to know whether there is a correlation between certain DNA types and antibiotic resistance (if it exists) as this information may identify M. pluton isolates "susceptible" to accepting resistance factors and identify areas where resistant may develop.

Industry Significance

European foulbrood (EFB) is a major cause of production losses to the beekeeping industry in Australia. The widespread nature of the disease in all States except Western Australia necessitated the introduction of antibiotic treatment in 1976 using oxytetracycline hydrochloride (OTC). OTC is the only antibiotic recommended for EFB treatment and although in place 21 years little is known regarding the current sensitivity of M. pluton to this antibiotic. There is, however, anecdotal evidence that OTC treatment is not always effective. This indicates that there is a need to determine the sensitivity of M. pluton so that beekeepers can be confident that treatment protocols are effective. If resistance of M. pluton to OTC has developed alternative control measures can then be pursued without unnecessary delay.

The eradication of EFB is unlikely due to its widespread nature and its ability to exist as a latent infection in hives. Hence, investigations of the organism to better understand its ability to develop resistance and to characterise isolates in terms of DNA profiles will provide State Departments of Agriculture and Industry with useful information which will guide them in future control strategy development. 

A literature search conducted by the library at this Institute has demonstrated that information in this area is lacking.

DNA restriction endonuclease profiles and typing of geographically diverse M. pluton isolates and their plasmid content will be determined using methodologies based on the work described in our previous project. This section of the project will provide information regarding the diversity of M. pluton in Australia and whether M. pluton has the ability to develop resistance via plasmids.

Following the examination of 49 isolates from a wide range of geographical areas it became clear that Australian M. pluton isolates belong to a very homogeneous group of organisms with insignificant variation in DNA restriction endonuclease profiles. All these isolates were also examined for the presence of plasmids. All isolates were plasmid-free.

In examining the brood samples submitted for this project Paenibacillus alvei was commonly encountered. Twenty eight isolates were examined. In direct contrast to M. pluton the P. alvei DNA restriction endonuclease profiles showed this group of organisms to be very diverse. A biochemical analysis of the reactions produced by these isolates also showed a great deal of diversity between isolates.

• To identify critical areas in queen bee production and introduction which may be contributing to low acceptance and poor early performance being reported with commercially reared queen bees

• To document current natural resources of the Queensland apiary industry by surveying and recording honey production, economic value and important floral species of apiary sites within Queensland. 
• Areas currently under utilised as bee forage areas to be possibly identified

The lack of documented and evaluated information on melliferous resources has restricted the apiary industry in arguing effectively to retain access to some valuable bee forage areas.

Data has been presented in formats able to be used by the beekeeping industry, and by Government Departments when matters concerning honeybees and land use requires discussion.

• New Guinea to mainland Australia, via the Torres Strait islands.
• To prevent the movement of Tropilaelaps clarae and Varroa jacobsoni mites from Papua

Our best prospect of keeping mainland Australia free of these mites is to maintain this separation of the two populations of honey bees.

However, there is a perception, among some islanders,that honey bees are essential to the production of food plants. In fact the bulk of traditional crops either do not require pollination agents, or are pollinated by native bees and/or other agents.

The benefits of the native bees in production of the fruits and crops have been promoted via various booklets and promotional material.

An activity book for school has been produced and distributed to Primary Schools in the Northern Peninsula Area (NPA) around Bamaga.. The book promoted the native (stingless) honey bees (Trigona spp and Austroplebeia spp.) as the local hero, "Trigon", fighting off the invading Asian honey bees, as well as other quarantine themes.

Heavy duty, plastic baggage tags featuring the "heroic" Trigon, produced in May 1996, have been distributed. Two thousand of these were produced. The label also features the Top Watch logo which was promoted by Mal Meninga in 1993.

A twelve page booklet detailing the life histories of selected native bees from The region has been produced. The text is illustrated with line drawings and a four page colour ‘center-fold’ shows some of the native bees, as well as a resting swarm of Asian honey bees. Two thousand booklets, plus an extra thousand copies of the colour insert, have been produced and are ready for distribution in the Torres strait, via the schools extension programme.

• Further investigate outbreaks of disappearing disorder in south east Queensland to define areas involved, seasonal occurrence and severity.
• Attempt to identify any particular floral resource that may be involved.
• Examine the possible role of heavy metals identified in pollens, and attempt to reproduce the disorder with a heavy metal feeding trial.
• Investigate if hive supplementary feeding can reduce or prevent disappearing disorder, and measure any production effects resulting from disappearing disorder.
• Investigate the possible role of flowering cat’s claw creeper with outbreaks of disappearing disorder.
• Attempt to transmit disappearing disorder with a frame of affected brood.

Experimental feeding, supplementary and transmission trials were undertaken at the Animal Research Institute, Yeerongpilly or at cooperating apiarist’s sites. The expertise of botanists, toxicologists, nutritional biochemists and microbiologists was recruited where necessary to support the investigations.

• Twenty six outbreaks of disappearing disorder were recorded during the two year period occurring from Rockhampton in central Queensland to Grafton in New South Wales. All cases were restricted to coastal areas. Outbreaks commenced after spring rains, and in one season extended to the following April, but this was reported as an abnormally long outbreak period. Cases varied from fleeting and mild, up to prolonged and severe, resulting in marked reductions in brood and adult bees.
• Botanical examination, and identification of pollens from affected larvae and hives, failed to incriminate any particular floral resource associated with disappearing disorder. The absence of cat’s claw pollen from all samples argued against the involvement of this suspected plant.
• Heavy metal examination of hive pollens revealed elevated levels of aluminium, copper, iron and zinc in several samples, but the levels were considerably lower and similar in affected and unaffected larvae. A feeding trail using aluminium and zinc in pollen patties produced a small number of affected larvae with aluminium patties at 200 mg/kg, but the results were not conclusive.
• The preventative value of supplementary feeding on disappearing disorder was also inconclusive, as disappearing disorder outbreaks did not occur in control hives at either site under observation. Supplementary feeding did not improve hive production when measured at one site.
• While outbreaks of disappearing disorder coincide with flowering of cat’s claw creeper, an attempted net feeding trial and location of hives adjacent to abundantly flowering creeper, failed to incriminate this plant.
• Transmission of disappearing disorder by inserting a frame of affected brood into a healthy hive could not be achieved. Microscopic examination of affected brood samples from outbreaks did not reveal any known brood pathogens.

• To survey the quality (chemical residues honeybee disease agents) of honey samples produced and packed in all states and territories in Australia.
• To examine the honeys for microbial quality (bacteria, yeasts and molds).
• To compare the results with those obtained in a previous report DAQ 202A Introduced Honeys – a Quality Survey.
• To research standard methodologies for assessing the microbial quality of honeys.
• To establish acceptable standards in terms of chemical residues bacteria, yeasts and molds for honey quality.

The further development of acceptable standards for honey quality will be a basis for industry to reassure consumers of the purity of honey. The ability of Australian honey producers to meet requirements in terms of residues, honeybee disease agents and microbial levels provides opportunities for increased national and international marketing.

All samples were cultured for honeybee disease agents namely American foulbrood (AFB) and chalkbrood. All honeys were also examined for microbial flora (bacteria, yeasts and molds) and for chemical residues.

Two separate diluent preparations were trialled to determine the most sensitive methods for assessing numbers of bacteria yeasts and molds.

• It is recommended that honey should contain <500 CFU’s/gram bacteria and a total of <10 CFU’s/gram yeasts and molds to be acceptable as a food product.
• This work provides useful information for honey packers who now have scientific information to guide their overall HACCP plans.

• Thirty one or 51.7% of honey samples screened contained either AFB or chalkbrood disease agents and returned greater then recommended numbers of bacteria yeasts and molds.
• A diluent preparation of peptone water with 40% glucose recovered greater numbers of yeasts and molds then peptone water diluent.
• High levels of lead were detected in 2 honey samples. Immediate action was taken to determine the sources and reason for this contamination. Investigation revealed that soldering in honey extracting equipment was responsible for leaching of lead into honey which was left in extracting equipment. Coating the soldering with food grade paint is believed to reduce the risk of this contamination.

• To develop and underpin honey bee nutrition with R, D & E proposals for further advances.

Fifteen workshop delegates discussed past practices, survey results, participant statements and quality assurance implications and recommended an eight point action plan.

Three 1998/99 mid-term projects are expected to be recommended to RIRDC.

Discussions are being held with Agriculture NSW regarding upgrading nutrition publications.

• To develop suitable management programs to control and reduce the level of AFB.

In 1993 the then industry peak body the Federal Council of Australian Apiarists’ Associations (FCAAA) decided it was time to setup an industry committee. FCAAA then asked the Animal Health Committee could it assist the industry to develop a national control program. The AHC set up a AHC working party to look at the issue. A face to face meeting took place of this committee in Sydney on October 1996 and a number of requests and suggestions were made to industry. From all this HBRDC set up a workshop to further develop the plans put forward by the AHC working party. The set of guidelines from the AHC working party became the agenda for the 29^th July 1998 workshop.

Industry will make the final decision.

• To have all beekeepers honey testing for AFB.
• To a national database program set up to maintain levels of AFB.
• To have uniform standards with the laboratories carrying out the honey testing and have these labs audited.
• To develop codes of practice for honey and queen producers.
• To develop a comprehensive quality assurance for beekeepers with packers. An essential part of this is a differential pricing for honey to encourage a quality product.
• Develop a standard national method to quantify the level of AFB that exists in the industry. If we do not develop a uniform statistical way of measuring the number of hives infected we can never show the industry is on top of it.
• The words amateur and commercial should be taken out, only refer to beekeepers.
• Australia should be divided into four zones when it comes to movements of bees/product etc. Western Australia, Kangaroo Island, Tasmania and the rest of Australia. Letting the first three to set their standards and the balance to be supported by vendor declarations, that is free movement.

• To determine if heat treatment can be used to sterilised beekeeping equipment that has been infected with Paenibacillus larvae subsp larvae, the spore-forming bacterium which causes American foulbrood (AFB) disease of honeybees.
• To examine the possibility of using powder-coating kiln to heat sterilise beekeeping equipment infected with spore of P. larvae subsp larvae.

A previous report (RIRDC Project QBA-1A) indicated that spores of P. larvae subsp larvae, can be destroyed by heat, but further work was required to accurately determine a temperature and time frame which would result in complete sterilisation of beekeeping equipment. This report also indicated that there were a number of variables affecting the ability of heat to destroy spores of P. larvae subsp larvae. These variables included heterogeneity of spores with respect to heat tolerance, time of exposure to heat, and the number of spores which may be present on infected equipment.

Laboratory Experiments

Spores from the pool were seeded on the surface of small pieces of pinewood and heated in a small laboratory oven. Exposures to dry heat at temperatures of 110⁰C and 130⁰C were trialled. At the conclusion of the heating regimes culture tests to determine the viability of the spores were performed.

Kiln Trial

A trial in a commercial powder coating kiln at Sumner Park in Brisbane was undertaken. An air hose was passed into the kiln to try to enhance the spread of heat through the heating chamber.

One hundred and four boxes were seeded with 3 x 10⁸ spores per site of P. larvae subsp larvae from the stock pool. Six additional boxes were seeded with spores and left out of the kiln to act as controls. The test boxes were exposed to dry heat at 130⁰C for 4 hours.

As for la boratory experiments the heated spores were harvested and cultured to determine viability.

The laboratory work showed that at 110⁰C, spores of P. larvae subsp larvae could be destroyed after exposure for 5 hours. At 130⁰C. the spores were killed after 3 hours exposure. These experiments were conducted under conditions of dry heat.

Commercial Kiln Trial

Powder coating plants are fairly common in cities and towns throughout Australia. It was reasoned that if the trials were to be us use to the beekeepers, there had to be premises that could be easily accessed. The powder coating plant could achieve the desired temperature of 130⁰C and hold for the time required, 4 hours. 

After heating at this time and temperature, there were 31 out of 104 sites (30%) which still showed viable spores of P. larvae subsp larvae.

• Cold spots near the floor of the chamber which did not reach the required temperature.
• Lack of super heated steam during the heating process.
• Time of heating was too short to be effective.

• To investigate the use of Australian liquid honey in commercial bakery products, and 
• To determine the effect of the incorporation of honey on the quality and functional properties of bakery products; 
• To communicate research findings to the Australian food processing industry.

Gelatinization of bread doughs: In the production of breads, it is important that gelatinization is not adversely affected. Too great an increase in the gelatinization temperature may yield a bread that is not fully baked in a standard oven, although, a small increase in gelatinization temperature is thought to slow the rate of staling. A previous RIRDC funded study suggested that adding honey to bread retards staling better than sugars alone.

Doughs with added honey were compared against doughs formulated using a model sugar solution and a control with no honey or sugar. Viscosity of batters: Batters were formulated using honey added at varying levels. Viscosity was then measured versus temperature using a visco-amylograph. Batters with added honey were compared against a control (0%) batter and batters containing a model sugar solution. Commercial optimization: Breads were made using a commercially available formulation with honey (manufacturing grade ironbark honey, courtesy of Capilano Honey Ltd.) at 2%, 3%, 4% and 6% levels. 

All treatments were compared against bread loaves incorporating comparable levels of a model sugar solution (the same sugars as the honey sample) as well as a control, containing no honey or model sugar solution. The effect of honey on the following properties were studied: dough proofing volume and time, crumb fineness and cellular elongation, crust thickness and contrast, and crumb texture (firmness) and water activity over a four-day period.

As these results were in model systems, it was necessary to apply this knowledge in a commercial bread formulation to ensure that the model system studies were valid. Here it was found that honey had little effect on the properties of the baking process. However, reanalysis of the results of an earlier study confirmed that adding honey at a level of 3% (w/w flour) significantly retarded the staling of white pan bread by at least 12 h relative to loaves containing 3% sugar and no sugar or honey.

There is a strong indication that the mode of action of honey is unique to the honey itself (ie. the minor components of the honey), rather than being due to the sugars of honey alone. This data will convince the Australian baking industry to seriously consider the use of honey at levels of up to 3%, since this level may retard staling, can not be detected when tasted (as sweetness), and does not adversely affect any stages of the baking process from dough development (mixing) to gelatinization.

2. Mackay, D.C., D’Arcy, B.R., Caffin, N.A. and Bhandari, B.R. Kinetics of gelatinization of plain bread doughs using honey. Cereals ‘97: Proceedings of the 47th Australian Cereal Chemistry Conference, Perth, 14-18 September 1997, 262-264. Melbourne, R Aust Chem Inst, 1997.

3. Squires, N., Ford, A., Nottingham, S. Caffin, N. and D’Arcy, B. The use of sensory evaluation to determine changes in bread texture during the staling process. Culinary Arts and Sciences II Global and National Perspectives, 411-419. Poole, UK, Worshipful Company of Cooks Centre for Culinary Research, 1998.

• The objectives of this project were 
• To find microorganisms, which are potentially antagonistic to Botrytis cinerea, and 
• To determine the effectiveness of honeybees to deliver biological control agents to plants susceptible to B. cinerea such as Geraldton waxflower.