Risk of European foulbrood in imported honey bee products

Transcription

1 Risk of European foulbrood in imported honey bee products Pharo, H.J. Team Manager Risk Analysis (Animal Kingdom), Biosecurity New Zealand, Ministry of Agriculture and Forestry, Wellington, New Zealand. Abstract New Zealand is free from a number of important diseases of honey bees, and the Ministry of Agriculture and Forestry has long been wary of the possibility of introducing these in imported honey bees, beekeeping equipment and bee products. Under the framework of the World Trade Organisation, the SPS agreement requires member countries to base their sanitary measures for imported animal products on a scientific assessment of risk. This paper presents the New Zealand experience with assessing the risk of introduction of honey bee diseases in imported honey bee products in particular, European foulbrood. Although heating is the sanitary measure most commonly applied to honey bee products for the inactivation of pathogens, for most diseases the scientific literature is incomplete concerning the degree of organism inactivation at different time/temperature combinations. New Zealand commissioned research on this for European foulbrood, and based the recommended measures on the results of that research. However, under the SPS agreement measures must be applied only to the extent necessary to achieve a country s acceptable level of risk, which requires countries to specify the acceptable level of risk. The absence of key scientific information limits the degree to which this can be done using quantitative analysis. Further research might assist decision-making, but the level of risk that is considered acceptable depends on stakeholder perceptions, and is unlikely to be unanimously agreed. Introduction The WTO and the SPS Agreement The World Trade Organisation framework consists of 60 agreements, annexes, decisions and understandings that are designed to provide an international forum to encourage free trade between member states, by regulating and reducing tariffs on traded goods and services, protecting intellectual property rights and providing a common mechanism for resolving trade disputes. One of the agreements under the WTO framework is the Agreement on the Application of Sanitary and Phytosanitary Measures, commonly known as the 'SPS Agreement'. This agreement, which came into force on 1 January 1995, sets out basic rules for food safety and animal and plant health standards in international trade. An SPS measure is a standard that is applied to an internationally traded product, and such measures often result in certain restrictions on trade. The SPS agreement stipulates that SPS measures may be applied only to the extent necessary to protect human, animal or plant health. However, governments may sometimes be pressured to go beyond what is needed for food safety and the protection of animal and plant health and to use SPS measures to shield domestic producers from economic competition. Safeguards that are not actually required for health reasons can be very effective protectionist devices, and because of their technical complexity, they can be deceptive and difficult to challenge (WTO, 1998). Therefore the key obligation on members under the SPS Agreement is that measures must not be applied in a way that would constitute a disguised restriction on trade. Members may apply those SPS measures that conform to international standards or others that are based on sound scientific assessment of the risks. In the case of international trade in animals and animal products, the relevant international standards are the guidelines recommended by the Paris-based World Organisation for Animal Health, the OIE (OIE, 2005).

2 The Import Risk Analysis Process The international guidelines for animals and animal products import risk analysis developed by the OIE (OIE, 2005), have been developed into detailed processes by the New Zealand Ministry of Agriculture and Forestry (MAF) as described in the MAF Risk Analysis Handbook (Murray, 2002). This process has been further refined by the OIE into two handbooks, one for qualitative risk analysis and one for quantitative risk analysis (OIE, 2004a; 2004b). The major steps in the MAF risk analysis process are as illustrated in Figure 1. These are: Hazard identification Risk assessment - Release assessment - Exposure assessment - Consequence assessment - Risk estimation Risk management In the hazard identification, the epidemiology or life history of each of the organisms of concern is examined so that a conclusion can be reached as to whether it should be regarded as a potential hazard in the context of the commodity under consideration. The release assessment evaluates in more detail each potential hazard in order to estimate the likelihood of the hazard being present in or on the commodity, and the likelihood of persistence of the hazard during processing, storage and transport. The release assessment thus provides an estimate of the likelihood of the hazard being associated with the commodity at the time of importation. The exposure assessment identifies biological pathways leading to exposure of susceptible hosts in New Zealand and estimates the likelihood that they will be exposed to the hazard. The consequence assessment describes the economic and health consequences associated with the exposure to the hazard, and the risk estimation step summarises the preceding three assessments to enable a decision to be made as to whether sanitary measures are necessary. Finally, risk management is the formulation of sanitary measures that are considered appropriate for the identified hazards. Underpinning the SPS agreement is an assumption that it is possible, through the application of rational analytical methods (scientific and economic), to objectively measure the levels of risk associated with proposed imports, to evaluate a number of options for risk management in terms of how much each option would reduce that risk, and to apply the measure or measures that delivered the correct (or appropriate) amount (or level) of protection (or risk reduction) in order to reduce the originally measured risk down to (but not below) a pre-determined acceptable level. Thus the 'appropriate level of protection' is closely related to the 'acceptable level of risk', as illustrated in Figure 2. In this hypothetical example, of the five measures available to manage the risk posed by the particular import, Measure 4 delivers the level of protection that is appropriate to reduce the risk to an acceptable level. The outcome of such a purely rational and scientific process would achieve the SPS goal of transparent management of biosecurity risk without unnecessarily restricting trade. In order to implement the above approach in its most literal sense, it is necessary not only to objectively assess the level of risk posed by a particular import proposal, but also to have established the acceptable level of risk for the disease agents of concern and the degree of risk reduction achievable by the available safeguards. The need for quantification in risk assessments is implicit in the SPS framework. However, as will be discussed here, scientific objectivity is difficult to achieve in practice, and quantification does not necessarily achieve it.

3 HAZARD IDENTIFICATION RISK ASSESSMENT Organisms of potential concern: * OIE listed * organisms affecting the economy, the people, the environment of New Zealand Is the organism associated with the animal species? concerned? No Not of concern in this risk analysis Release assessment How likely is the agent to be introduced in the commodity? Yes No Is the organism likely to be associated with the commodity? Yes Exposure assessment How likely are susceptible animals to be exposed? Is the organism exotic to New Zealand? Yes Not considered to be a potential hazard in this commodity No Are strain differences reported in other countries? Yes Potential hazard in the commodity Consequence assessment What are the likely consequences of exposure? No No Is there a control programme in New Zealand? Yes Risk estimation Is the organism considered to be a hazard in the commodity? RISK MANAGEMENT What is the acceptable level of risk? How does the assessed risk compare to the acceptable level of risk? Yes No What safeguards are available? What is the effect of each safeguard on the level of risk? Apply safeguards that reduce risk from assessed level to acceptable level No safeguards necessary Figure 1 The MAF risk analysis process Risk of European Foulbrood in Honey The first MAF risk analysis to examine the likelihood of introduction of disease agents in imported honey bee products was completed in Concerns were raised during public consultation that some areas of the risk analysis were inadequate, and as a result MAF carried out a further risk analysis that was released for public consultation in late Both of these analyses were qualitative. The commodities considered in the 2004 risk analysis were honey, royal jelly, pollen, propolis, beeswax and bee venom. The following discussion on the risk of European foulbrood in imported honey is based on that risk analysis.

4 12 Measures that are available to reduce the risk associated with this particular import from the level of risk that is assessed towards the level of risk that is acceptable. Measure 1 Measure 2 Measure 3 Measure 4 Measure 5 10 Level of risk (in 'risk units') This is the amount of protection that is appropriate in this case. 2 0 Assessed Acceptable Figure 2 Risk management, and the relationship between the acceptable level of risk and the appropriate level of protection (Source: Pharo, 2003). Hazard Identification European foulbrood (EFB) is a disease of larvae of Apis mellifera, Apis cerana and Apis laboriosa caused by a fastidious anaerobic bacterium, Melissococcus pluton (Bailey and Collins, 1982a, 1982b). Larvae become infected by being fed contaminated brood food by nurse bees. The bacteria begin to grow vigorously within the midgut, and by the time the larva is 5 days old, the area of the midgut that is normally occupied by the food mass is packed by the bacteria, creating an abnormal demand for food. If there are too many young larvae for the available brood food nurse bees normally eject such larvae because they are the first to show signs of starvation. If the population of nurse bees is such that surplus food is available, infected larvae can survive and in this way most colonies can keep the infection contained (Shimanuki, 1997). Infected larvae that survive discharge M. pluton bacteria with larval faeces onto the wall of the brood cells (Bailey, 1959b), where they appear to survive better in the thin smears than in dead larval remains. Most M. pluton are removed from cells by house-cleaning worker bees, which then act as passive vectors to contaminate larval food, but it appears that M. pluton does not grow in the gut of adult bees (Bailey, 1957), probably because of the relatively aerobic conditions in that environment. Clinical symptoms of EFB are only likely when the ratio of nurse bees to diseased larvae decreases for some reason, such as when nurse bees are recruited away from larval feeding by the demands of a high nectar flow. When this imbalance occurs, infected larvae that have a higher than normal demand for food are not removed and visual signs of the disease in the form of diseased larvae in combs begin to appear (Alippi, 1999). Once sufficient nurse bees are again able to clean out dead larvae, the disease usually subsides (Bailey and Ball, 1991). Therefore, honey bee colonies are usually more seriously affected during the spring and early summer (Tarr, 1938; White, 1920). In experiments carried out early in the 20th century, M. pluton was shown to survive for about 12 months in an incubator and at normal room and outdoor temperatures. In honey exposed to direct sunlight the organism was destroyed after 3-4 hours, while in honey stored away from direct sunlight the organism survived up to 7 months (White 1920). In view of the above, M. pluton is classified as a potential hazard in imported honey.

5 Release Assessment It is generally accepted that M. pluton can be present in honey, as well as in pollen, beeswax, propolis and royal jelly (OIE, 2005). M. pluton has been reported in honey from infected hives, albeit relatively infrequently. In one study, 6% of bulk honey samples from endemic areas were culture positive for M. pluton (Hornitzky and Smith, 1998). The polymerase chain reaction (PCR) assay appears to be more sensitive than culture for detecting M. pluton in honey a study of 80 honey samples from different states in Australia found 22/80 (28%) positive by culture but 57/80 (71%) positive using PCR (McKee et al, 2003). Only one report exists in the scientific literature on the concentration of M. pluton in honey under natural conditions. Wootton et al (1981) reported that examination of honeys from a limited number of honeybee colonies infected with EFB in New South Wales Australia contained up to 3.3 x 10 3 culturable M. pluton organisms per ml (3.5 log 10 per ml). However, bacterial culture for M. pluton is an insensitive test as only about 0.2% of the organisms detected by microscopy in honey are culturable (Hornitzky and Smith, 1998). Moreover, the relationship between culturability and infectivity is not known for this organism. Thus, the normal level of infectious M. pluton organisms in honey under natural conditions is uncertain, but it may be considerably higher than the level reported by Wootton et al (1981). M. pluton is a fastidious anaerobic bacterium (Bailey, 1959b), and the high sugar concentration and presence of natural bacterial inhibitors in honey probably prevent its growth in honey. However, the duration of survival of this organism in honey is unknown, notwithstanding that it is a hardy organism that is known to survive for long periods in the environment, for example up to 15 months in smears on glass slides (Bailey, 1959a). Since M. pluton has been demonstrated to be present in unprocessed honey, the likelihood of release is considered to be non-negligible. Exposure Assessment For M. pluton bacteria in imported honey bee products to come into contact with susceptible species in an importing country, the imported products would have to be harvested by worker bees and taken back to hives and fed to young larvae, or be fed directly to the colony by beekeepers. Honey is highly attractive to bees, and research commissioned by MAF suggests that a honey concentration above 2% is likely to make manufactured or processed products attractive to bees (Goodwin and Cox, 2004). The scientific literature contains no information on the infectious dose of M. pluton under natural conditions, and only one report on this under experimental conditions. In this experiment, McKee et al (2004) extracted M. pluton organisms from infected larvae and fed these organisms to artificially reared larvae at various concentrations, as determined by haemocytometer. The lowest concentration of M. pluton in larval food that led to any larval mortality was 200 organisms per ml. When larval food containing 2 x 10 5 organisms per ml was fed to larvae continuously, a linear relationship was observed between the duration of feeding the contaminated food and the larval mortality. The regression equation calculated by McKee et al (2004) (r 2 = 0.92) was: mortality (%) = 0.62 (hours of feeding) 2.86 Using this equation, a dose-response relationship may be constructed for these particular experimental conditions, as shown in Table 1.

6 Table 1 Relationship between larval mortality and duration of feeding larvae with 2 x 10 5 M. pluton organisms/ml (McKee et al, 2004). Mortality % Hours of feeding It is important to note that in the experiments of McKee et al (2004) the natural housekeeping effect of adult bees in the hive was absent. Therefore, it is not possible to extrapolate from these experimental results to natural conditions, except to say that the infectious dose of M. pluton is likely to be considerably higher under natural conditions. Nevertheless, even in the highly artificial environment of this experiment, prolonged feeding of contaminated food was necessary to transmit EFB to larvae. Since the infectious dose of M. pluton is not known under natural conditions, the likelihood of exposure cannot be objectively determined. Nevertheless, the available information supports the conclusion the likelihood of exposure of M. pluton in imported honey to susceptible species in an importing country is non-negligible. Consequence Assessment In the presence of uninterrupted nectar flows the level of infection with M. pluton usually remains low, in which case colonies can cope with the infection without assistance (Shimanuki, 1997; Alippi, 1999). However, under some circumstances involving limited nectar flows in spring and early summer honey bee colonies may be destroyed or crippled by European foulbrood (Bailey and Ball, 1991). The disease has been reported to be a problem in bee colonies used for pollination (Shimanuki, 1997), which has potential impacts on pastoral farming, crops and fruit. Moreover, in many countries where EFB is present, beekeepers find it necessary to feed antibiotics to control disease, which may result in negative trade effects for exports of honey bee products. In view of the variable effect of EFB under natural conditions, predicting the rate and extent of spread and the impact at local and national levels if it were introduced into a country is not feasible. Therefore, quantification of the likely consequences is not possible, and all that can be concluded is that experience in other countries suggests that the introduction of this disease is likely to result in negative effects on pollination, losses in production of bee products, disease control costs for beekeepers through the need to feed antibiotics, and testing and certification costs to honey exporters in regard to antibiotic residues. These potential consequences are considered to be significant, and the consequence assessment is therefore non-negligible. Risk Estimation Although the limited information on the level of M. pluton organisms likely to be present in honey and lack of information on the infectious dose for larvae means that the likelihood of release and exposure cannot be accurately calculated, it is concluded that both are non-negligible. Since the consequences of introduction are also non-negligible, the risk of M. pluton is considered to be nonnegligible and the organism is considered to be a hazard in honey.

7 Risk Management Risk evaluation Since the risk estimate for EFB is considered to be non-negligible, risk management measures are justified for these commodities to reduce the risks to an acceptable level. Option evaluation The OIE Terrestrial Animal Health Code (the Code) (OIE, 2005) recommends that honey from countries that are not free from European foulbrood should have been processed to ensure the destruction of M. pluton. However, the Code does not specify what treatments are considered appropriate for the destruction of M. pluton *. The options available are heat (alone or in combination with hydrostatic pressure) or gamma irradiation. In addition to these treatments, it may be possible to test imported honey for the presence of M. pluton. Heat A study from the USA in the early 1900s estimated the thermal death point of M. pluton suspended in honey to be 79 C / 10 minutes (White, 1914). This estimate was based on the ability to infect honey bee larvae. However, further trials were subsequently carried out in Australia in 1980 (Wootton et al, 1981) and in the UK in 2001 (Ball et al, 2001), resulted in further estimates based on bacterial culture, as shown in Table 2. These estimates were considerably more cautious for 80 C the inactivation time was estimated by Wootton (1981) to be an hour, while Ball et al (2001) estimated it to be 17 minutes. The thermal death times estimated by Wootton et al (1981) also suggested variation between the five different honeys tested the value shown in Table 2 is the maximum value. It was suggested that different ph and water content of the honeys might be responsible for the different thermal death times observed, but no simple relationship was apparent (Wootton et al, 1981). This phenomenon has not been further investigated. Ball et al (2001) carried out further experiments over a wider range of temperatures and fitted a first order kinetic model to the data, assuming a linear relationship between the logarithm of the number of survivors and time. The linear regression model of log 10 (cfu + 1) against time for each temperature treatment, was used to predict D values (i.e. time for one log 10 reduction in organism count, or in other words the time for a 90% reduction) and extinction times. These calculated values are shown in Table 2. In addition to the temperatures shown in the table, Ball et al (2001) included treatments at temperatures of 90 C and 100 C in their experiments, but when fitting regression lines and determining D values, the data for these two higher temperatures were not used because inactivation was so rapid (somewhere between 2 and 5 minutes) that insufficient data points were available for those temperature. The effect of honey type on thermal inactivation of M. pluton is incompletely understood. Wootton et al (1981) used 5 different honeys each with a single predominant floral source, and found considerable variation between honeys, but no variation within each honey type. Ball et al (2001) used a single blended honey derived from several European countries. Nevertheless, the estimates of thermal death point in the these two studies are in reasonable agreement for temperatures C. * In the 14 th edition of the Terrestrial Animal Health Code, 2005, article refers to measures that are in Appendix XXX (under study). Thermal death point is defined in food processing terminology as the temperature at which an organism dies in a given time. The thermal death time is the time it takes for an organism to die at a given temperature.

8 Table 2. Thermal inactivation of M. pluton in honey results of two studies. Wootton et al, 1981 Ball et al, 2001 Temperature ( C) Maximum thermal death time Extinction time h 47 h 30 min 60 8 h 12 h 38 min 70 3 h 30 min 1 h 53 min 80 1 h 17 min In order to explore the assumption of linearity and to overcome a mathematical difficulty associated with the calculation of extinction time used by Ball et al (2001), Cox and Domijan (2004) applied an alternative empirical fit model to the data, allowing the development of a predictive model that allows any D value to be calculated for any temperature. The values shown in Table 3 were produced using that model. Although the Cox and Domijan (2004) model does allow the prediction of required treatment times for temperatures above 80 C, the authors are in agreement with Ball et al (2001) that the limited number of data points above that temperature result in uncertainty in predicted times, and this reservation is borne out by the wider confidence intervals for those predictions. Table 3 Inactivation time in minutes - comparison of predictions from two models using the data of Ball et al (2001). Ball et al (2001) Cox and Domijan (2004) Temperature ( C) D value (minutes) Extinction time (min) 1D value (min) 4D value (min) 6D value (min) < Note that : 1D reduction is a 90% reduction in the concentration of organisms 4D reduction is a 99.99% reduction in concentration of organisms 6D reduction is a % reduction in concentration of organisms Heat and hydrostatic pressure A sterilisation process has been developed in the USA that uses a combination of hydrostatic pressure and temperature to inactivate spores of Clostridium botulinum in honey used for baby food (Omahen, 2004). Standard US industry practice for pasteurisation of honey is heating to 76 C and holding it for about 5 minutes. This is adequate to destroy fungi and yeasts, but is not adequate for bacterial spores such as those of C. botulinum these require heating to at least 120 C under pressure and holding at this temperature for at least 3 minutes. However, heating honey to such high temperatures causes unacceptable changes to its flavour and texture which this new sterilisation process is designed to overcome. The process pressurises honey to about 2,400 bar (35,000 psi) and passes it very quickly through a heat exchanger to raise its temperature to 82 C within a few

9 seconds. When the pressure is dropped, the temperature instantly spikes to about 135 C, after which the honey is cooled within seconds. Since bacterial spore survival at that high temperature is measured in seconds, the result is a sterile product that has not been physically changed. Researchers at the University of Georgia have patented this process and are about to evaluate it for the inactivation of honey bee pathogens in extracted honey (Toledo, personal communication ). It is possible that such processes might be commercially applicable in the future. Gamma irradiation The use of gamma radiation to destroy M. pluton has been investigated in a number of different commodities (Hornitzky, 1981, 1994). Although 0.8 Mrad (800,000 rad, or 8kGy) was insufficient to kill M. pluton (Pankiw et al, 1970), 1.0 to 1.5 Mrad (10 15 kgy) was used to eliminate the organism from honey (Hornitzky, 1981). In honey that had a starting concentration of 1.23 x 10 5 organisms per ml, no organisms survived 14 kgy, and this appears to be a generally recommended treatment level (Hornitzky, 1994). Although irradiation of honey may result in a slight intensification of flavour as well as a lightening of colour (Katznelson and Robb, 1962), it is considered that it causes no significant deterioration of honeys as measured by colour and taste (Hornitzky, 1994) Testing for the presence of M. pluton in bee products Despite its limited sensitivity, bacterial culture has been used to detect M. pluton in a range of bee products (Bailey, 1984; Giacon and Mallone, 1995; Hornitzky and Smith, 1998). The polymerase chain reaction (PCR) assay can also be used for the detection of M. pluton in honey (Djordjevic et al, 1998; Govan et al, 1998; McKee et al, 2003). However, the use of PCR for testing honey that has been heat-treated to kill M. pluton may be of questionable value, as PCR can detect the DNA of dead organisms. Recommended sanitary measures The risk analysis recommended that for the management of the risk of EFB in honey, each consignment of imported honey must be : Discussion (i) from a country or part of the territory of a country free from European foulbrood or (ii) gamma irradiated with 15 kgy or (iii) Heated to achieve a 6D reduction in organism numbers according the model of Cox and Domijan (2004), using agitation to ensure the even distribution of heat and automatic temperature tracing to demonstrate that the core temperature has been reached before timing begins. Due to uncertainty and variability, quantitative analysis is not necessarily more objective nor more defensible than qualitative analysis. Comprehensive quantitative risk analysis is rarely possible, and most analyses contain both qualitative and quantitative elements. This assessment of the risk posed by M. pluton in imported honey emphasises the difficulty of applying the quantitative risk assessment framework envisaged under the SPS agreement. Although in this instance the degree of risk reduction achievable by risk management measures has been established with unusual accuracy Dr Romeo Toledo, Food Scientist, College of Agricultural and Environmental Sciences, University of Georgia.

10 by the commissioning of specific research, the inability to precisely measure release, exposure and consequences thwart a purely quantitative assessment. Biosecurity decision makers do not have the luxury of postponing import risk decisions until there is perfect scientific information. Decisions must be made as to whether goods can be safely traded or not, given current scientific information and currently available risk management measures. WTO member countries are obliged under the SPS agreement to apply risk management measures consistently, only to the extent necessary, and without being overly restrictive on trade. Although the OIE has recognised that there is a non-negligible risk of EFB in honey bee products, and that treatment to destroy M. pluton is appropriate, there is currently no international standard specifying what that treatment should be. The question therefore becomes one for individual decision makers. Time/temperature treatments can be specified to result in different levels of confidence that M. pluton is destroyed. A 6D reduction is a million-fold or % reduction in the concentration of organisms present. Due to the different calculation methods used in the respective studies, however, how this level of protection relates to that achieved by the irradiation safeguard is unknown. Opinions differ between stakeholder groups as to how cautious decision makers should be in the face of imperfect scientific information. In this instance, scientific uncertainty regarding the level of M. pluton in honey under natural conditions and the infectious dose for this organism has resulted in different perceptions of the risk and different views on the acceptability of whatever risk remains after risk management measures are applied. Although the 6D reduction is a commonly applied principle in human food safety, this is a relatively risk-averse position to take for animal products. However it is not inconsistent with the level of protection (4D to 5D) achieved by the international standards for heat treatment of milk products from countries or zones with foot and mouth disease (Donaldson, 1997). Under the SPS agreement member countries do have the right to adopt provisional measures if there is insufficient scientific evidence to demonstrate that trade can occur safely. However, such provisional measures may only be imposed for a reasonable time while additional information is sought. It was under this principle that New Zealand maintained a prohibition on honey imports for more than a decade while research into the heat inactivation of M. pluton was carried out, culminating with the report of Ball et al (2001). It would be theoretically possible to continue that prohibition while the other two areas of uncertainty identified above are further investigated. However, although surveys on the level of M. pluton normally found in raw honey could be carried out relatively easily in, a standard measure of organism concentration would first have to be established before further information in this matter could be useful. Further, conducting further research on the infectious dose of M. pluton for honey bee colonies would be more problematic, as noted by McKee et al (2004), due to the sporadic and variable infection results that have been experienced in this kind of work to date (see for example Bailey, 1960). Given the different interests of stakeholders, there is no reason to suspect that further research would resolve the differences of opinion about the acceptability of the risk posed by imported honey. Rather, the likely outcome would be to shift the focus of contention to the limitations of the new research. Groups that are opposed to imports are unlikely to ever be convinced that the level of protection offered by the chosen risk management measures is adequate, just as groups in favour of imports do not agree that the risk warrants management beyond certain limits. Scientific uncertainty is a fact of life, and unanimous agreement among stakeholders and scientists is rarely possible. Zero risk is not attainable, whether trade occurs or not, and the acceptability of any particular import risk depends on stakeholder perceptions as to the distribution of the benefits from the trade in question. Thus, acceptable risk decisions are essentially political judgements that attempt to reflect societal values, and such decisions can at best be informed by science without being purely scientific.

11 References Alippi, A.M. (1999) Bacterial diseases. In: Colin ME, Ball BV, Kilani M (eds). Bee Disease Diagnosis Pp Centre International de Hautes Etudes Agronomiques Mediterraneennes, Zaragoza. Bailey, L. (1957) The isolation and cultural characteristics of Streptococcus pluton and further observations on Bacterium eurydice. Journal of General Microbiology 17, Bailey, L. (1959a) An improved method for the isolation of Streptococcus pluton and observations on its distribution and ecology. Journal of Insect Pathology 1, Bailey, L. (1959b) Recent research on the natural history of European foulbrood disease. Bee World 40, Bailey, L. (1960) The epizootiology of European foulbrood of the larval honey bee, Apis mellifera Linnaeus. Journal of Insect Pathology 1, Bailey, L. (1984) A strain of Melissococcus pluton cultivable on chemically defined media. Federation of European Microbiological Societies Microbiology Letters 25, Bailey, L. and Ball, B.V. (1991) Honey Bee Pathology. Academic Press, London. Bailey, L. and Collins, M.D. (1982a) Taxonomic studies on Streptococcus pluton. Journal of Applied Bacteriology 53, Bailey, L. and Collins, M.D. (1982b) Reclassification of Streptococcus pluton (White) in a new genus Melissococcus, as Melissococcus pluton nom. rev.; comb. nov. Journal of Applied Bacteriology 53, Ball, B.V., Wilson, J.K., Clark, S. (2001) Determination of the thermal death time of Melissococcus pluton in honey. IACR-Rothamsted, UK, 27 pp. Unpublished report to MAF Biosecurity Authority, New Zealand. Cox, N. and Domijan, K. (2004) Modelling thermal destruction of viruses and bacterial cells. Unpublished report to MAF Biosecurity on the analysis of data of Ball et al (2001). Donaldson, A.I. (1997) Risks of spreading foot and mouth disease through milk and dairy products. OIE Scientific and Technical Review 16(1), Djordjevic, S.P., Noone, K., Smith, L.A., Hornitzky, M.A.Z. (1998) Development of a hemi-nested PCR assay for the specifc detection of Melissococcus pluton. Journal of Apicultural Research 37, Giacon,H. and Malone, L. (1995) Testing imported bee products for European foulbrood. New Zealand Beekeeper 2(8): 8-9. Goodwin, M. and Cox, H. (2004) Attractiveness of a range of sugars and honey bee products to honey bees. Report to MAF Biosecurity dated September Hortresearch client report no s : and

12 Govan, V.A., Brozel, V., Allsopp, M.H., Davison, S. (1998) A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae. Applied Environmental Microbiology 64, Hornitzky, M.A.Z. (1981) Use of gamma radiation from cobalt 60 in the control of Streptococcus pluton in honey. Bee World 75, 135- Hornitzky, M.A.Z. (1994) Commercial use of gamma radiation in the beekeeping industry. Bee World 75, Hornitzky, M.A.Z. and Smith, L. (1998) Procedures for the culture of Melissococcus pluton from diseased brood and bulk honey samples. Journal of Apicultural Research 37, Katznelson, H. and Robb, J.A. (1962) The use of gamma radiation from cobalt-60 in the control of diseases of the honeybee and the sterilization of honey. Canadian Journal of Microbiology 8, McKee, B., Djordjevic, S.P., Goodman, R.D., Hornitzky, M.A.Z. (2003) The detection of Melissococcus plutonius in honey bees (Apis mellifera) and their products using a hemi-nested PCR. Apidologie 34: McKee, B.A., Goodman, R.D., Hornitzky, M.A. (2004) The transmission of European foulbrood (Melissococcus plutonius) to artificially reared honey bee larvae (Apis mellifera). Journal of Apicultural Research 43 (3): Murray, N. (2002) Import Risk Analysis: Animals and Animal Products. Wellington, New Zealand Ministry of Agriculture and Forestry. OIE (2004a) Handbook on Import Risk Analysis for Animals an Animal Products. Volume 1, introduction and qualitative risk analysis. OIE (World Organisation for Animal Health), Paris, France. OIE (2004b) Handbook on Import Risk Analysis for Animals an Animal Products. Volume 2, quantitative risk assessment. OIE (World Organisation for Animal Health), Paris, France. OIE (2005) Terrestrial Animal Health Code. 14 th Edition. World Organisation for Animal Health, Paris, France. Omahen, S. (2004) It s safe, honey! Columns, November 4, University of Georgia. Pankiw, P., Bailey, L., Gochnauer, T.A., Hamilton, H.A. (1970) Disinfection of honeybee combs by gamma irradiation. II: European foulbrood disease. Journal of Apicultural Research 9(3), Pharo, H.J. (2003) The impact of new epidemiological information on a risk analysis for the introduction of avian influenza viruses in imported poultry meat. Proceedings of Fifth International Symposium on Avian Influenza, April 14-17, 2002, University of Georgia, Athens, GA. Avian Diseases 47, Shimanuki, H. (1997) Bacteria. In: Morse R, Flottum K (eds). Honey Bee Pests, Predators, and Diseases Third Edition. Pp AI Root, Ohio.

13 Tarr, H.L.A. (1938) Studies on European foulbrood of bees. IV. On the attempted cultivation of Bacillus pluton, the susceptibility of individual larvae to inoculation with this organism and its localisation within its host. Annals of Applied Biology 25, White, G.F. (1914) Destruction of germs of infectious bee diseases by heating. US Department of Agriculture Bulletin 92, 8 pp. May 15, White, G.F. (1920) European Foulbrood. US Department of Agriculture Bulletin 810. Wootton, M., Hornitzky, M., Ryland, L. (1981) Thermal Destruction of Streptococcus pluton in Australian Honeys and Its Effect on Honey Quality. Journal of Apicultural Research 20, WTO (1998) Understanding the WTO Agreement on Sanitary and Phytosanitary Measures. World Trade Organisation website: Acknowledgments Brenda Ball, Rothamstead, UK Leone Basher, Biosecurity New Zealand Neil Cox, AgReasearch, New Zealand Paul Bolger, Biosecurity New Zealand Katie Owen, Biosecurity New Zealand Michael Hortnitzky, Department of Primary Industries, NSW, Australia