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SUMMARY
The causal organism of European foulbrood of honey bees is the bacterium Melissococcus pluton. The identification of its presence by the observation of signs of disease in the field is unreliable. The most usual and obvious sign is the death of larvae shortly before they are due to be sealed in their cells, but this may be for reasons other than European foulbrood. Most infected colonies display few visible signs, which themselves often quickly abate spontaneously before the end of each active season. Infection remains enzootic within individual colonies because of mechanical contamination of the honeycombs by the durable organism. Recurrences of disease can therefore be expected in subsequent years.
Identification of the agent: Examination, by high-power microscopy, of suitable preparations of larval remains for the presence of numerous lanceolate cocci is adequate for most practical purposes, especially when it is done by experienced individuals.
The only certain means of making a diagnosis is by isolating and identifying the causative organism. This can be differentiated quite readily from all other bacteria associated with bees by its fastidious cultural requirements.
The isolated bacterium can be identified and differentiated by means of simple tube agglutination tests.
Serological tests: No tests for detecting antibodies in bees are available.
Requirements for vaccines and diagnostic biologicals: There are no biological products available.
A. INTRODUCTION
Bee larvae usually die of European foulbrood 1-2 days before being sealed in their cells, or sometimes shortly afterwards, and always before transformation to pupae. The disease is caused by Melissococcus pluton and occurs mostly during the period when colonies are growing quickly. Most sick larvae become displaced from their coiled position in the bottom of their cells before they die. Many are quickly detected and removed by nurse bees, leaving empty cells scattered randomly among the remaining brood.
Infected larvae that escape detection by adult bees and then die, first become flaccid and turn a light yellow colour that becomes increasingly brown, and at the same time they dissolve into a semi-liquid mass. They then become dry and form a dark brown scale that can easily be removed from the cells. Severely affected brood may have a very stale or sour odour, sometimes acidic, like vinegar, but often there is no smell.
Signs of disease usually disappear spontaneously from infected colonies before the end of the active season, but are likely to return in subsequent years (3, 10).
B. DIAGNOSTIC TECHNIQUES
| 1. | Identification of the agent
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| | a) | Microscopy
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| | | Freshly dead larvae are best for diagnosis. Before any decomposition occurs, diseased larvae can be smeared on a microscope slide or pulled apart by pinching the cuticle about the centre of the body with two pairs of forceps, which are then pulled apart. The mid-gut contents are left exposed on the slide, still within the gelatinous, transparent peritrophic membrane. This is partially or almost completely filled with bacteria, which are easily seen as opaque chalk-white clumps. The contents of the mid-guts of healthy larvae, which are less easily dissected, have a golden-brown colour. Apparently healthy larvae may contain a mixture of bacteria and pollen. The mid-guts of healthy larvae that contain much light-coloured pollen may resemble those that are filled with bacteria.
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Fig. 1. Bacteria associated with European foulbrood.
(a) Melissococcus pluton: The cause of European foulbrood. Occurs singly, in longitudinal chains or in clusters. Morphologically resembles Enterococcus faecalis, a common secondary invader.
(b) Paenibacillus alvei: Vegetative rods 2.0-7.0 ( 0.8-1.2 µm with flagella; sporulating with spores lying adjacently. Both rods and spores are larger than those of Paenibacillus larvae (see American foulbrood).
(c) Bacterium eurydice: Slender, square-ended rods in vivo but can form chains of cocci in vitro
in certain media.
| | | For a bacteriological investigation, a loopful of a dilute aqueous suspension of the mid-gut contents is transferred to a clean microscope slide and mixed with a loopful of 5% aqueous nigrosin. This is spread over one or two square centimetres, dried gently over a flame, and examined directly by high-power microscopy. The presence of numerous lanceolate cocci, about 0.5 x 1.0 µm in size, occurring either singly or in clusters, and arranged end to end in pairs or short chains, is almost certainly diagnostic of European foulbrood. Some very slender square-ended rod-like bacteria are also usually present (Figure 1). The cocci are Gram positive and the rods are Gram negative. Similar preparations made from aqueous suspensions of whole dead or decomposing larvae are likely to present a confusing array of bacteria in which M. pluton will be difficult to distinguish.
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| | b) | Culture methods
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| | | Melissococcus pluton (type strain NCDO 2443) is the most abundant bacterium during the early stages of an infection (4, 5). Melissococcus pluton can be cultivated on a medium (expressed in g/litre or ml/litre) comprising: yeast extract or certain peptones, 10 (4); cysteine or cystine, 0.2-2.0; glucose or fructose, 10; soluble starch, 10; 1 M KH2PO4, 100 at pH 6.6; and agar, 2. The medium is preferably autoclaved in 100 ml lots in screw-capped bottles at 116°C for 20 minutes and poured into Petri plates immediately before use. These plates are streaked with dilute aqueous suspensions of diseased larvae, or ideally, of diseased larval mid-guts. The latter can be prepared beforehand by allowing them to dry on a slide, which may then be kept, for years if necessary, at 4°C or -20°C. All culture media should be subjected to quality control and must support the growth of M. pluton from small inocula. The reference strain should also be cultured in parallel with the suspect samples to ensure that the tests are working correctly.
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The preparation and storage of dried smears also eliminates most secondary organisms after a few weeks without affecting the viability of M. pluton. This organism is isolated most efficiently by inoculating decimal dilutions of the aqueous suspension into agar that has been maintained molten at 45°C and which is then poured into plates. The plates must be incubated anaerobically, such as in McIntosh and Fildes jars in an atmosphere of approximately 5-10% carbon dioxide (CO2) at 35°C. Small white opaque colonies of M. pluton usually appear within 4 days. This bacterium is somewhat pleomorphic in vitro, often appearing in rod-like forms. The final pH of the medium may reach 5.5. Decreasingly fastidious strains become selected in vitro. Simplified or modified forms of the medium then support multiplication, especially of a serologically distinct M. pluton group from Brazil (1) that will multiply on chemically defined media (2). CO2 remains essential. Inoculated slopes should be sealed when bacterial growth is apparent and may then be kept at 4°C for up to 6 months. Alternatively, the cultures can be suspended in a medium of 10% sucrose, 5% yeast extract and 0.1 M KH2PO4, pH 6.6, and then lyophilised.
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| | | A number of other bacteria are often associated with and may be confused with M. pluton. Bacterium eurydice inhabits the alimentary tract of adult bees and occurs commonly in the gut of healthy larvae in small numbers. It is more numerous in larvae infected with M. pluton. The incidence of B. eurydice in healthy bees is very low in winter and early spring, but it increases in summer. It forms thin square-ended rods, which can grow either singly or in chains. When grown in certain media, it sometimes resembles streptococci and has been confused with M. pluton. However, its cultural characteristics closely resemble those of Corynebacterium pyogenes (10), and it multiplies poorly in the form of thin rods, under the conditions necessary for the cultivation of M. pluton.
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| | | Enterococcus (= Streptococcus) faecalis closely resembles M. pluton morphologically and has often been confused with it, although they are both culturally and serologically distinct. Unlike M. pluton, it does not remain viable for long when dried, or persist as mechanical contamination within bee colonies. It is probably brought into the hive by foraging adult bees, and is responsible for the sour smell sometimes encountered with European foulbrood.
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Enterococcus faecalis grows well in vitro under the conditions suitable for M. pluton, but it may be readily differentiated by its ability to grow aerobically. It forms small transparent colonies within 24 hours and is a facultative anaerobe. It multiplies on a variety of the more common media with or without carbohydrates or CO2. The final pH in the presence of glucose is 4.0. Enterococcus faecalis rarely exceeds the number of M. pluton in bee larvae, and can usually be diluted out. When it is not diluted out it produces sufficient acid to prevent the in-vitro multiplication of M. pluton.
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| | | Enterococcus faecalis does not multiply in bee larvae in the absence of M. pluton, so its presence in large numbers can be taken as presumptive evidence of European foulbrood.
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| | | Paenibacillus (= Bacillus) alvei is generally more common than E. faecalis in bee colonies affected with European foulbrood, but it is not invariably associated with the disease and so cannot act as a reliable indicator of it. In bee colonies, it multiplies only in the decomposing remains of larvae, and then its spores often predominate over all other bacteria, even to their apparent exclusion. Paenibacillus alvei forms very resistant spores and becomes well established in bee colonies with enzootic European foulbrood. It causes a characteristic stale odour. Paenibacillus alvei multiplies poorly under the conditions necessary for the in-vitro growth of M. pluton. It produces a spreading growth of transparent colonies, some of which are motile and move in arcs over the surface of the agar. Cultures have the characteristic stale odour that is associated with European foulbrood when the bacillus is present. Spores are formed rapidly.
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| | c) | Immunological methods
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| | | For the identification of M. pluton, antisera can be prepared in rabbits against washed cultures of M. pluton either by intravenous injections (6) or by a single intramuscular injection of 1 ml of antigen suspension mixed with an equal volume of Freund's incomplete adjuvant.
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| | | Assays are made by agglutination tests in tubes containing suspensions of bacteria equivalent to 0.25 mg dry weight/ml. End-points are noted after tubes have been incubated for 4 hours at 37°C.
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| | d) | Nucleic acid recognition methods
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| | | Detection of M. pluton by polymerase chain reaction has been reported (7, 9). Specific primers to regions of the 16S rRNA gene have been identified, but the technique is not yet widely used. DNA profiles of M. pluton have been described and DNA restriction endonuclease (Alu1) analyses could be used to reveal genotypic diversity among geographically diverse isolates of M. pluton (8).
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| 2. | Serological tests
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| | No tests for detecting antibodies in bees are available.
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C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
There are no biological products available.
ACKNOWLEDGMENT
Illustrations by Karl Weiss, extracted from Bienen-Pathologie, 1984. Reproduced with the kind permission of the author and Ehrenwirth-Verlag, Munich (Germany).
REFERENCES
| 1. | Allen M.F. & Ball B.V. (1993). The cultural characteristics and serological relationships of isolates of Melissococcus pluton. J. Apic. Res., 32, 80-88.
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| 2. | Bailey L. (1984). A strain of Melissococcus pluton cultivable on chemically defined media. FEMS Microbiol. Lett., 25, 139-141.
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| 3. | Bailey L. & Ball B.V. (1991). Honey Bee Pathology. Academic Press, London, UK, and New York, USA.
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| 4. | Bailey L. & Collins M.D. (1982). Taxonomic studies on Streptococcus pluton. J. Appl. Bacteriol., 53, 209-213.
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| 5. | Bailey L. & Collins M.D. (1982). Reclassification of Streptococcus pluton (White) in a new genus Melissococcus, as Melissococcus pluton nom. rev.; Comb. nov. J. Appl. Bacteriol., 53, 215-217.
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| 6. | Bailey L. & Gibbs A.J. (1962). Cultural characters of Streptococcus pluton and its differentiation from associated enterococci. J. Gen. Microbiol., 28, 385-391.
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| 7. | Djordjevic S.P., Noone K., Smith L. & Hornitzky M.A.Z. (1998). Development of a semi-nested PCR assay for the specific detection of Melissococcus pluton. J. Apic. Res., 37, 165-174.
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| 8. | Djordjevic S.P., Smith L.A., Forbes W.A. & Hornitzky M.A. (1999). Geographically diverse Australian isolates of Melissococcus pluton exhibit minimal genotypic diversity by restriction endonuclease analysis. FEMS Microbiol. Lett., 173, 311-318.
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| 9. | Govan V.A., Brozel V., Allsopp M.H. & Davison S. (1998). A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae. Appl. Environ. Microbiol., 64, 1983-1985.
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| 10. | Jones D. (1975). A numerical taxonomic study of Coryneform and related bacteria. J. Gen. Microbiol., 87, 52-96.
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NB: There are OIE Reference Laboratories for Bee diseases (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).
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