EXAMINATION OF HONEY AND ADULT BEES FOR EARLY DETECTION OF PAENIBACILLUS LARVAE LARVAE

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1 EXAMINATION OF HONEY AND ADULT BEES FOR EARLY DETECTION OF PAENIBACILLUS LARVAE LARVAE Abstract Ingemar Fries, Sanna Nordström Department of Entomology, Swedish University of Agricultural Sciences Box 7044, Uppsala, Sweden Tel: , Fax: , .ingemar.friea@entom.slu.se,.susann.nordstrom@entom.slu.se In apiaries with clinical symptoms of American foulbrood (AFB) in some honey bee colonies, or with a history of AFB in the apiary, samples of honey (30 g) and adult bees (100 bees) have been taken from the brood room. Collected samples have been analyzed for Paenibacillus larvae larvae using MYPGP agar incubated in 5% CO 2. In colonies without symptoms of American foulbrood there were significantly more samples of bees that tested positive for P. larvae larvae compared to samples of honey from the same colonies. Furthermore, the average number of germinating spores were higher in bee samples compared to honey samples. In colonies with symptoms of AFB the results also demonstrate that more viable spores of the pathogen is retrieved by using bee samples compared to honey samples. The presented results demonstrate that if the objective is to find P. larvae larvae in individual honey bee colonies, samples of bees from the brood room are more sensitive than the use of honey samples collected from the same area of the hive. Introduction American foulbrood in honey bees (Apis mellifera) is caused by the sporeforming bacteria Paenibacillus larvae larvae (formely Bacillus larvae (White, 1907). The infective spores enter the digestive tract of young larvae where they germinate and the rods enter the body cavity and proliferate in the haemolymph (Ratnieks, 1992). As the infected larvae succumb to the infection, the bacterium sporulates again and large numbers of extremely environmentally stable spores are produced that retain their infectivity for decades (Bailey and Ball, 1991). Detection of infection of American foulbrood in the field is based on the appearance of infected brood. Dead larvae turn brown and expose a characteristic ropy consistency and cappings over such larvae sink inwards and become sunken and dark colored. As the bees begin to remove dead brood the cappings become perforated. However, investigations of honey has demonstrated that the disease may persist endemically in infected colonies for several years without any visible symptoms (Hansen and Rasmussen, 1986). Hansen (1984a) developed a method for detection of American foulbrood spores in honey based on direct inoculation of diluted honey on J-agar (Gordon et al., 1973) after thermal treatment. This method has been used with good results in a number of studies (Hansen, 1984b; Hansen and Rasmussen, 1986; Steinkraus and Morse, 1992). Several investigators have altered the method by changing medium, adding a centrifuging step or including antibiotics in the medium (Alippi, 1995; Hornitzky and Clark, 1991; Lloyd, 1986; Ohe, 1997; Ritter and Kiefer, 1993; Shimanuki and Knox, Proceedings of the 37 th International Apicultural Congress, 28 October 1 November 2001, Durban, South Africa APIMONDIA 2001 To be referenced as: Proc. 37 th Int. Apic. Congr., 28 Oct 1 Nov 2001, Durban, South Africa ISBN: Produced by: Document Transformation Technologies Organised by: Conference Planners

2 1988; Steinkraus and Morse, 1992). Although J-agar is useful for isolation of P. larvae larvae compared to some media used (Hornitzky and Nicholls, 1993), MYPGP agar (Dingman and Stahly, 1983) has proven better in a number of comparisons (Nordström and Fries, 1995; Ritter and Kiefer, 1993; Steinkraus and Morse, 1996). Addition of CO 2 in the incubation environment also stimulates sporulation and growth of the bacterium (Hornitzky and Nicholls, 1993; Nordström and Fries, 1995). Nordström & Fries (1995) improved the direct inoculation technique by Hansen (1984a) by changing the medium to MYPGPagar and incubating the plates in 5% CO 2, thus, making the test significantly more sensitive. However, irrespective of whether bulk samples from entire apiaries are used (Kabay, 1995), or if samples are collected from individual colonies (Nordström et al., 1997), a few honey samples may produce false negative results. In other words, honey samples may be false negative even if the apiary, or the individiual colony, contains visual symptoms of AFB. Hornitzky (1988, 1989) isolated P. larvae larvae on brain heart infusion agar and incubation in air from adult bees in clinically diseased colonies and demonstrated that all bee samples from such colonies were positive for the bacterium. From 77 samples of bees from colonies without symptoms, but with AFB present in the apiary, 23 were positive (Hornitzky and Karlovskis, 1989). This method has later been used to study spread of AFB between colonies (Goodwin et al., 1994b), the presence of AFB spores in feral colonies (Goodwin et al., 1994a) and as an instrument to locate clinically diseased colonies without actual inspection (Goodwin et al., 1996). When bees are used for detection of P. larvae, bees from the brood room is to be preferred (Goodwin et al., 1996). To detect subclinical levels of AFB may prove useful for studies of the spread of this disease and could be used to determine where prohylactic measures may prevent disease outbreak. Although samples of honey as well as bees can be used to detect subclinical levels of P. larvae spores in honey bee colonies, there are no records available where these type of samples have been compared. We have taken samples of both bees and honey from diseased and visually healthy colonies, in apiaries where AFB has been detected and compared the ability of both sample types to find P. larvae larvae spores in honey bee colonies. Material and Methods Inspectors from the National Bee inspector Program in Sweden were asked to collect samples when apiaries with visual symptoms of American foulbrood were detected. Within a year from detection of AFB, samples of bees from the brood room and honey adjacent to the brood were collected both from clinically diseased colonies and colonies without symptoms of disease. A total of 13 samples of both bees and honey from colonies with symptoms of AFB and a total of 164 samples from visually healthy colonies from apiaries where the disease had been recently detected, were analyzed. Honey samples were processed as described by Nordström & Fries (1995). Five grams of each honey sample were diluted with the same amount of water and heated in glass bottles for 10 minutes in a water bath with a temperature of 90 C. After heating, one loopful of each honey solution was inoculated on 6 MYPGP agar plates using a disposable 10 µl inoculation needle (Difco) and incubated in 5% CO 2 for 7 days when number of colonies of P. larvae were counted. With colony counts >100 the value 100 was recorded. Confirmation of P. larvae larvae was based on colony morphology, rod morphology, spore production of the bacterium, and a negative katalase test. Samples of 100 bees each were put into a nylon mesh (1.2 x 1.2 mm) bag. The mesh bag was then introduced into a 1 liter plastic bag and sealed with a heat sealer. Twenty ml of a sterile NaCl in water solution (9mg/ml) were added before closing the bag. The bees in the bag were crushed by

3 rolling a bottle with some pressure over the flattened bag and the resulting fluid was collected. The collected fluid was centrifuged at g for 10 minutes and the resulting pellet dissolved in 2 ml of the sterile NaCl solution. This fluid was heated in glass bottles for 10 minutes in a water bath with a temperature of 85 C and inoculated, incubated and analyzed as described for the honey samples. Results In colonies without symptoms of American foulbrood (N=164), there were significantly more samples of bees that tested positive for P. larvae compared to samples of honey from the same colonies (p<0.001, chi-square=8.97, 1 df). Furthermore, the average number of colonies were higher in bee samples compared to honey samples from the same bee colonies (p<0.01, paired t- test, 163 df). The data for clinically healthy colonies is tabulated in Table 1. In colonies with symptoms of AFB (N=13) the results also demonstrate that with the methods used, more viable spores of the pathogen is retrieved by using bee samples as described compared to honey samples. Furthermore, in 1 sample of honey from colonies with clinical symptoms of AFB, the test failed to demonstrate presence of viable spores. In the colonies with symptoms of disease, all bee samples demonstrated presence of the pathogen and a larger proportion of these samples scored the maximum count compared to samples of honey (12 and 4 respectively; p<0.001, chisquare=15,48, 1 df). The plating data for clinically disesed colonies is tabulated in Table 1. Table 1. Distribution of samples positive (+) and negative (-) for Paenibacillus larvae larvae in samples of honey and bees from the same colonies. Also given is the average number of bacterial colonies per inoculated plate (Colonies per plate) and the standard error (SE) of this average. Note that the maximum number of colonies recorded is 100, i.e underestimating colony numbers in sample types (in this case bees) where the maximum number is more comon Clinical symptoms of AFB No clinical symptoms of AFB Honey Bees Honey Bees Number of Samples Colonies per plate SE Colonies per plate N N

4 Discussion The presented results demonstrate that if the objective is to find P. larvae larvae in individual honey bee colonies, samples of bees from the brood room, processed as described, are more sensitive than the use of honey samples collected from the same area of the hive and analyzed as described by Nordström & Fries (1995). The method described for samples of bees is based on direct inoculation after a centrifuging step which increases the amount of work needed compared to the honey method. The use of disposable material, such as plastic bags eliminates risks of cross contamination of samples. There are more contaminants when bee samples are used compared to honey samples for detection of P. larvae larvae (data not shown). This may reflect the slightly lower temperature used in heating the bee samples compared to the honey samples. The lower temperature for the bee samples have been used because spores of P. larvae larvae are more sensitive to heating in water compared to heating spores in a 1:1 solution of honey and water (data not shown). However, from the Swedish samples we have investigated, contaminants are a minor problem. In a few samples it was necessary to redo the test because of contaminants, but we have not seen the amount of contamination reported elsewhere from honey (Alippi, 1995), not even in the bee samples. Generally, the swarming Bacillus alvei seem to be the most common problem for isolation of P. larvae larvae (Hornitzky and Clark, 1991). Addition of 3µg/ml nalidixic acid to the growth medium has then proved successful (Hornitzky and Clark, 1991). It seems logical that samples of live bees should represent the present health status of sampled colonies better than samples of honey. Stored honey, sealed as well as unsealed, may have entered the hive when there were no disease in the colony. This could explain why honey samples may be negative, even in colonies with clinical symptoms of disease. Honey may also be robbed from diseased colonies and produce samples positive for P. larvae larvae without actually producing disease in the sampled colony. Obviously, samples of bees will detect more infected colonies compared to honey samples. It should be further investigated if the spore levels in samples of bees can be used to determine the relative risk of detecting clinical symptoms in honey bee colonies. It shouls also be investigated if composite samples from entire apiaries can be used as a prognostic tool for detection of American foulbrood.

5 References Alippi A.M. (1995) Detection of Bacillus larvae spores in Argentinian honey using a semi-selective medium, Microbiología SEM 11, Bailey L., Ball B.V. (1991) Honey bee patholohgy, Academic Press, London Dingman D.W., Stahly D.P. (1983) Medium promoting sporulation of Bacillus larvae and metabolism of medium components, Appl. Environ. Microbiol. 46, Goodwin R.M., Houten T.A., Perry J.H. (1994) Incidence of American foulbrood infections in feral honey bee colonies in New Zealand, New Zealand Journal of Zoology 21, Goodwin R.M., Perry J.H., Haine H.M. (1996) A study of the presence of Bacillus larvae spores carried by adult honey bees to identify colonies with clinical symptoms of American foulbrood disease, Journal of Apicultural Research 35, Goodwin R.M., Perry J.H., Ten-Houten A. (1994b) The effect of drifting honey bees on the spread of American foulbrood infections, Journal of Apicultural Research 33, Gordon R.E., Haynes W.C., Pang C.H. (1973) The Genus Bacillus, United States Department of Agriculture, Washington, D. C. Hansen H. (1984a) The incidence of the foulbrood bacterium Bacillus larvae in honeys retailed in Denmark, Danish Journal for Plant and Soil Science 88, Hansen H. (1984b) Methods for determining the presence of the foulbrood bacterium Bacillus larvae in honey, Danish Journal for Plant and Soil Science 88, Hansen H., Rasmussen B. (1986) The investigation of honey from bee colonies for Bacillus larvae, Danish Journal for Plant and Soil Science 90, Hornitzky M.A.Z., Clark S. (1991) Culture of Bacillus larvae from bulk honey samples for the detection of American foulbrood, Journal of Apicultural Research 30, Hornitzky M.A.Z., Karlovskis S. (1989) A culture technique for the detection of Bacillus larvae in honeybees, Journal of Apicultural Research. 28, Hornitzky M.A.Z., Nicholls P.J. (1993) J-medium is superior to sheep blood agar and brain heart infusion agar for the isolation of Bacillus larvae from honey samples, Journal of Apicultural Research 32, Kabay M.J. (1995) Evaluation of the culture of honey to detect American foul brood, Australian Veterinary Journal 72, Lloyd J.M. (1986) Simplified laboratory diagnosis of American foulbrood disease, Journal of Apicultural Research 25, Nordström S., Forsgren E., Fries I. (1997) Amerikansk yngelröta - hur effektivt är det att sanera hela bigården?, Bitidningen 96, Nordström S., Fries I. (1995) A comparison of media and cultural conditions for identification of Bacillus larvae in honey, Journal of Apicultural Research 34, Ohe v.d., W. (1997) Efficient prophylactic measures against American foulbrood by bacteriological analysis of honey for spore contamination, American Bee Journal 137, Ratnieks F.L.W. (1992) American Foulbrood: the spread and control of an important disease of the honey bee, Bee World 73, Ritter W., Kiefer B. (1993) Eine Methode zum Nachweis von Bacillus larvae in Honigproben, Tierärztliche Umschau 48, Shimanuki H., Knox D.A. (1988) Improved method for the detection of Bacillus larvae spores in honey, American Bee Journal 28, Steinkraus K.H., Morse R.A. (1992) American foulbrood incidence in some US and Canadian honeys, Apidologie 23, Steinkraus K.H., Morse R.A. (1996) Media for detection of Bacillus larvae in honey, Acta Biotechnol. 16, White G.F. (1907) The cause of American foul brood, USDA, Bureau of Entomology Circular 94, 4 pp

6 EXAMINATION OF HONEY AND ADULT BEES FOR EARLY DETECTION OF PAENIBACILLUS LARVAE LARVAE Ingemar Fries, Sanna Nordström Department of Entomology, Swedish University of Agricultural Sciences Box 7044, Uppsala, Sweden Tel: , Fax: , .ingemar.friea@entom.slu.se,.susann.nordstrom@entom.slu.se Date of Birth: June 29, 1950 Education: Curriculum vitae: Ingemar Fries Doctor of Agronomy, March 1988, Swedish University of Agricultural Sciences, Uppsala Thesis: Contribution to the study of nosema disease (Nosema apis Z.) in honey bee (Apis mellifera L.) colonies. Employment: Associate Professor, Department of Entomology, Swedish University of Agricultural Sciences, Uppsala Research leader, Bee Division, Swedish University of Agricultural Sciences, Uppsala Visiting Fellow, Entomology Department, Cornell University, Ithaca, NY, USA Coordinator of the Concerted Action Integrated control of varroa mites (Contract no FAIR CT ) with the European Commission with 15 European partners. Responsible for the Swedish bee disease laboratory in Uppsala Recent selected Publications Fries, I., Camazine, S Implications of horizontal and vertical pathogen transmission for honey bee epidemiology. Apidologie 32, in print. Fries, I., Perez-Escala, S Mortality of varroa mites (Varroa destructor) inhoney bee (Apis mellifera) colonies during winter. Apidologie 32, in print. Nordström, S., Ball, B., Fries, I Influence of larval rearing condition and Varroa jacobsoni infestation on prevalence of deformed wing virus infection and wing deformity in newly emerged honey bees. Journal of Invertebrate Pathology, accepted. Calis, J.N.M., Fries, I., Ryrie, S Population modelling of Varroa jacobsoni Oud. Apidologie: 30, Fries, I Epizootiology of honey bee diseases in cold climates. Proc Apimondia 36th International Apicultural Congress. Vancouver, Canada, Fries, I., Paxton, R.J., Tengö, J., da Silva, J.A., Slemenda, S.B., Pieniazek, N.J Morphological and molecular characterization of Antonospora scoticae n. gen., n. sp. (Protozoa, Microsporidia) a parasite of the communal bee, Andrena scotica Perkins, 1916 (Hymenoptera, Andrenidae). European Journal of Protistology: 35, Nordström, S., Fries, I., Aarhus, A., Hansen, H., Korpela, S Virus infections in Nordic honey bee colonies with no, low, or severe Varroa jacobsoni infestations. Apidologie 30, Major research interests: Microsporidia biology and taxonomy Varroa mite biology and control Honey bee epidemiology