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SUMMARY
American foulbrood affects the larval stage of the honey bee Apis mellifera and other Apis spp., and occurs throughout the world. Paenibacillus larvae subsp. larvae (White), the causative organism, is a bacterium that can produce over one billion spores in each infected larva. The spores are extremely resistant to heat and chemical agents, and only the spores are capable of inducing the disease.
Identification of the agent: Combs of infected colonies have a mottled appearance due to a mixture of healthy capped brood, uncapped cells containing the remains of diseased larvae, and empty cells. This is not characteristic only of American foulbrood. Cell cappings of a diseased larva appear moist and darkened, becoming concave and possibly punctured as infection progresses. The larval or pupal colour changes to creamy brown and then to a dark brown with a ropy appearance when drawn out. A distinctive odour develops in the advanced stage. The diseased brood eventually dries out to form characteristic brittle scales that adhere tightly to the lower sides of the cell. The formation of a pupal tongue is one of the most characteristic, but rarely seen, signs of the disease and precedes the formation of the scales.
The method of choice for diagnosis of American foulbrood depends on whether clinical signs of the disease are present or not. In case of clinical illness, a range of simple confirmational laboratory techniques are available. Some of them require the isolation of the pathogenic agent by subculturing. The presence of heat-resistant spores, growth characteristics of the bacterium, colony morphology, combined with the following simple laboratory tests are considered to be conclusive: Gram staining, catalase test and nitrate reductase test (facultative). Thorough identification of the isolated bacteria can be done by biochemical profiling or polymerase chain reaction (PCR). The latter also permits direct examination of the larval remains without the previous long cultivation step. Antibody-based techniques are useful when no cross-reactivity with other bacilli has been demonstrated, for instance against Paenibacillus alvei, often found in late phase European foulbrood.
When clinical signs are absent or information on the appearance of the brood combs are missing (examination of honey bee products) a thorough identification of the pathogenic agent is recommended. This can be done by biochemical profiling (on suspicious isolated colonies) or by PCR (directly on the samples or after cultivation). Only experienced persons can rely on growth characteristics, colony morphology, and the above-mentioned simple confirmational laboratory techniques alone.
Serological tests: There are no serological tests available.
Requirements for vaccines and diagnostic biologicals: There are no biological products available.
A. INTRODUCTION
American foulbrood is a disease of the larval stage of the honey bee Apis mellifera and other Apis spp., and occurs throughout the world where such bees are kept. Paenibacillus larvae subsp. larvae (White), the causative organism, is a bacterium that can produce over one billion spores in each infected larva. The bacterium is a round-ended, straight and sometimes curved rod, which varies greatly in size (0.5 µm wide by 1.5-6 µm long), occurring singly and in chains and filaments; some strains are motile. The sporangia are often sparse in vitro, and the ellipsoidal, central to subterminal spores, which may swell the sporangia, are often found free (15). The spores are extremely heat stable and resistant to chemical agents. Only spores are capable of inducing the disease.
The infection can be transmitted to a larva by nurse bees or by spores remaining at the base of a brood cell. Although the larval stages of worker bees, drones and queens are susceptible to infection, infected queens and drone larvae are rarely seen under natural conditions. The susceptibility of larvae to American foulbrood disease decreases with increasing age (32); larvae cannot be infected later than 53 hours after the egg has hatched. The mean infective dose (ID50= spore dose at which 50% of the larvae are killed) needed to initiate infection, though very variable, is 8.49 spores in 24-48-hour old bee larvae (13). Exchanging combs containing the remains of diseased larvae is the most common way of spreading the disease from colony to colony. In addition, feeding or robbing of spore-laden honey, artificial swarms and the introduction of queens from infected colonies can also spread the disease. The early detection of American foulbrood helps to prevent further spread.
B. DIAGNOSTIC TECHNIQUES
| 1. | Identification of the agent
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| | A healthy larva has a glistening, pearly white appearance. It first develops at the base of the cell in the shape of the letter 'C' and subsequently grows upright to fill the cell. Infected larvae die in this erect position. In severely infected colonies, the combs appear to be mottled due to a pattern of healthy capped brood, uncapped cells containing the remains of diseased larvae, and empty cells. The capping of a cell that contains a diseased larva appears moist and darkened and becomes concave and punctured as the infection progresses. Also, the larva or pupa changes colour, first to a creamy and eventually to a dark brown. The larvae become glutinous in consistency and can be drawn out as threads when a probe is inserted into the larval remains and removed from the cell. A distinctive odour develops at this stage, resembling that of animal glue. Finally, after 1 month or more, the remains of the diseased brood dry out to form typical hard, dark scales that are brittle and adhere strongly to the lower sides of the cell (Figure 1). If death occurs in the pupal stage, the formation of the pupal tongue, a protrusion from the pupal head that traverses the top of the brood cell, is one of the most characteristic signs of the disease, although it is rarely seen (Figure 2c). The tongue may persist also on the dried scale.
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Fig. 1. Progression of the disease: (a) Point of infection. (b) Larval development to the prepupal stage.
(c) Cell contents reduced and capping is drawn inwards or is punctured. (d) Cell contents become glutinous.
(e) Residual scale tightly adherent to bottom of cell.
| | The method of choice for diagnosis of American foulbrood depends on whether clinical signs of the disease are present or not. In case of clinical illness, a range of simple confirmational laboratory techniques are available. Some of them require the isolation of the pathogenic agent by subculturing. The presence of heat-resistant spores, growth characteristics of the bacterium, colony morphology, combined with the following simple laboratory tests are considered to be conclusive: Gram staining, catalase test and nitrate reductase test (facultative). Thorough identification of the isolated bacteria can be done by biochemical profiling or polymerase chain reaction (PCR). The latter also permits direct examination of the larval remains without the previous long cultivation step. Other methods for direct examination of larval remains are described: the modified hanging-drop technique, the Holst milk test and different antibody-based techniques. The modified hanging-drop technique, based on the Brownian movements of P. l. larvae spores and their morphology, is poorly specific. The same is true for the Holst milk test, based on a high level of proteolytic activity during sporulation of P. l. larvae. Both tests should not be used alone. Antibody-based techniques are useful when no cross-reactivity with other bacilli has been demonstrated, for instance against Paenibacillus alvei, often found in late phase European foulbrood.
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| | When clinical signs are absent or information on the appearance of the brood combs is missing (examination of honey bee products) a thorough identification of the pathogenic agent is recommended. This can be done by biochemical profiling (on suspicious isolated colonies) or by PCR (directly on the samples or after cultivation). Only experienced persons can rely on growth characteristics, colony morphology, and the above-mentioned simple confirmational laboratory techniques alone.
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Fig. 2. Clinical American foulbrood (a-c) and Gram staining (d): (a) Combs have mottled appearance.
(b) A matchstick draws out the brown, semi-fluid larval remains in a ropy thread. (c) The formation of a pupal tongue is a very characteristic sign, but rarely seen. (d) Microscopic examination of isolated colonies reveals Gram-positive rods, occurring singly and in chains.
| | a) | Sample preparation
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| | | When the typical signs of disease are observed at the apiary it is recommended to send to the laboratory a piece of brood comb of about 20 cm2, containing as much of the dead or discoloured brood as possible. Little or no honey should be present in the sample. The sample can be loosely wrapped in paper, and wrappings, such as plastic bags, aluminium foil, waxed paper, tin or glass, should be avoided as these materials allow samples to become mouldy, making an accurate diagnosis almost impossible. The sample can be dispatched in a heavy cardboard or wooden box. If a portion of comb cannot be sent, the probe used to examine cell contents must have enough material on it for any test. This too can be wrapped in paper or put into an appropriate tube. However, such a small sampling size can only be considered when the sampler has sufficient expertise or is well trained to identify the diseased areas on the comb.
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| | | Sometimes, the larval remains are difficult to locate because of the condition of the comb. Scale material can be conveniently located by using ultra-violet or near ultra-violet light. Exposure between 310 and 400 nm will cause any scale material to fluoresce. Some discretion must be used when using this technique as both honey and pollen will also fluoresce.
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| | | When macroscopic examination at the apiary by an experienced sampler reveals that the brood combs have a healthy appearance, honey samples may be sent for laboratory analysis. Samples of food supplies collected from sealed cells close to the brood nest can be taken with a spoon and transferred into a plastic bag or tube (31). Harvested honey ready for sale can be taken as well, although this does not enable the identification of diseased colonies if there are spores present in the sample. Samples size should be 30-50 g.
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| | b) | Culture techniques
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| | | To culture P. l. larvae from larval remains, spore suspensions are prepared by mixing diseased material in 5-10 ml of sterile water, physiological solution (phosphate buffered saline or 0.9% NaCl) or liquid medium (same composition as the solid media listed below, but without agar) in a test tube. All culture media should be subjected to quality control and must support the growth of P. l. larvae 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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| | | Honey samples to be examined for spores are heated to 45-50°C and shaken to distribute any spores that may be present. Dilution with an equal volume (25 ml) of water permits easier handling. The diluted honey is transferred into 44 mm width dialysis tubing that has been tied at one end. The open end is tied after filling. The tubes are submerged in running water for 18 hours or in a water bath with 3-4 water changes over the same time period. After dialysis, the contents are centrifuged at 2000 g for 20 minutes. The supernatant liquid is discarded leaving approximately 1 ml (or less) of residue in each sample. The deposit is then resuspended in 9 ml of water (29).
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| | | Honey can also be prepared for culturing without the dialysis step, however this requires longer (30 minutes) and faster centrifugation (3000 g). Likewise, the volume in which the deposit finally is resuspended can be kept much smaller (200 µl) in order to improve the sensitivity of the test (6). Whatever the method of choice will be, when the outcome of honey analyses is done in a quantitative way and threshold values are set, the methodology that was used to establish these values should always be strictly followed.
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| | | Both the sample preparations derived from brood comb samples and honey samples can be processed in the same way from this point on. The suspension is heated at 80°C for 10 minutes to kill nonsporulating bacteria. A sterile cotton swab is used to transfer a portion of the suspension on to the surface of Petri dishes containing solid medium, which are then incubated for 2-4 days at
34-37°C. For a quantitative evaluation, it is recommended to spread a fixed volume of the suspension on solid agar with a sterile scraper rather than using cotton swabs. Inoculated plates are best incubated in an atmosphere of 5-10% CO2 in air, although aerobic incubation will do as well.
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| | | Different solid media can be used including brain-heart infusion agar supplemented with thiamine HCl (29), J-medium (19), MYPGP (7), Michael's medium (5) and Columbia agar containing 5% horse blood (15). The latter may become discoloured or partially haemolysed when P. l. larvae grows on it.
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| | | Samples from clinically diseased larvae will result in confluently grown plates after 2-4 days, leading to a subculturing step in order to have isolated colonies. On Columbia blood agar, colonies are small (< 1 mm in diameter), regular, glossy, butyrous, and greyish or discoloured with blood pigments (15). On Michael's medium, the colonies are whitish, opaque, flattened, with irregular edges and usually with a diameter of 1-3 mm (5). Inexperienced technicians are advised to run P. l. larvae reference strains in parallel, for instance LMG 9820 (other designation: ATCC 9545). A proven positive brood or honey sample might serve as a positive control for the entire examination.
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| | | Difficulties can occur when the spore suspensions contain other spore-forming bacteria, which may completely overgrow the culture. If so, it is recommended to supplement the solid medium with nalidixic acid (18) and/or pipemidic acid (2). Stock solutions are prepared by dissolving 0.3 g nalidixic acid or 0.4 g pipemidic acid in 2 ml 1 N NaOH and diluting to 100 ml with 0.01 M phosphate buffer or water, sterilised by filtration and stored under refrigeration. These stock solutions are added to molten agar medium to give a final concentration of 6-9 µg/ml nalidixic acid and 10-20 µg/ml pipemidic acid.
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| | | Spores similar to P. l. larvae have also been recovered from bees wax by chloroform extraction (21) and from pollen by an aquaeous filtration (10).
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| | c) | Simple confirmational tests
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| | | Paenibacillus l. larvae is a Gram-positive rod with some characteristics that make it distinguishable from many other bacilli that contaminate bees and bee products. Indeed, the bacterium is catalase negative (14) and reduces nitrate to nitrite (23). In consequence, a number of simple laboratory tests can confirm American foulbrood if clinical signs are observed and when cultivation of heat-treated samples yields colonies with the characterised growth velocity (slow) and colony morphology (see above) containing Gram-positive rods.
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| | | Catalase test: a drop of 3% hydrogen peroxide is placed on an actively growing culture on solid medium. Most aerobic bacteria break down the peroxide to water and oxygen, producing a bubbly foam, but P. l. larvae is almost always negative for this reaction (14). When Colombia blood agar is used for cultivation, the test cannot be done on the solid medium as the presence of horse blood will cause a false-positive reaction. In this case, colonies should be transferred to a clean microscope slide for the execution of the test.
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| | | Nitrate reductase test: the bacterium can be grown on a medium such as brain-heart infusion agar containing potassium nitrate (1-2 mg/litres of medium). When growth has occurred, the addition of a drop of sulphanilic acid-alpha-naphthyl reagent produces a red colour if any nitrate has been reduced to nitrite. Nitrate reductase negative P. l. larvae strains have also been described (16, 20).
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| | d) | Biochemical profile
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| | | When the analyst cannot rely on the presence of clinical signs (for instance, examination of honey bee products) or when the disease is still in its subclinical phase, a more profound identification of the pathogenic agent is recommended. Biochemical profiling of suspicious isolated colonies, in addition to their basic characteristics (heat-resistance, growth velocity, colony morphology and bacterium morphology) can be considered to be conclusive. The biochemical profiling includes - besides the above-mentioned catalase and nitrate reductase tests - the production of acid from carbohydrates, the hydrolysis of starch and casein, the use of citrate and the liquefaction of gelatin.
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| | | Production of acid from carbohydrates (11): bacteria are grown in J-broth (same composition as J-medium, but without agar) in which 0.5% of the test substrate, separately sterilised in aqueous solution, is substituted for the glucose. The carbohydrates used are L (+)-arabinose, D (+)-glucose, D (+)-xylose and D (+)-trehalose. The cultures are tested at 14 days by aseptically removing 1 ml or less to a spot plate, mixing the sample with a drop of 0.04% alcoholic bromocresol purple, and observing the colour of the indicator. Paenibacillus l. larvae produces acid aerobically from glucose and trehalose. No acid is produced from arabinose and xylose (1). Differentiation between P. l. larvae and P. l. pulvifaciens - the latter associated with the rare disease named 'powdery scale' - can be done based on acid production from mannitol and salicin (15). This seems to be one of the few characteristics that allow differentiation at the subspecies level. In addition, P. l. pulvifaciens also grows at 20°C (P. l. larvae does not) and some strains produce yellow/ orange-pigmented colonies (15).
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| | | Hydrolysis of starch (11): 1 g of potato starch is suspended in 10 ml of cold distilled water, mixed with 100 ml of J-medium without glucose, autoclaved, cooled to 45°C, and then thoroughly mixed and poured into five Petri dishes. After 3 days' storage at room temperature (to allow the excess moisture to evaporate), duplicate plates are streaked with each culture. The plates are flooded with Gram's iodine after 5 and 10 days' incubation. After 15-30 minutes, the unchanged starch becomes white and opaque. A clear zone underneath (after the growth was scraped off) and around the growth, measures the hydrolysis of the starch. Paenibacillus l. larvae strains do not hydrolyse starch.
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| | | Hydrolysis of casein (5): the medium that is used for this test is composed of a solution A (10 g skim milk powder, 90 ml distilled water) and a solution B (3 g agar, 97 ml distilled water) that are sterilised separately (121°C for 20 minutes), brought to 45°C in a double boiler and then mixed. The medium thus prepared (25 ml) is poured into Petri dishes, which are inoculated with 24-hour cultures. Incubation is continued for 7 days. A positive reaction is indicated by clarification of the medium under and around the colony growth area. Paenibacillus l. larvae causes a positive reaction.
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| | | Use of citrate: this is tested using semisolid J-medium without glucose but supplemented with 2 g of sodium citrate (11). The medium, sterilised in test tubes, is inoculated with two to three drops of a young (3-4 days) culture in semisolid J-medium. After 14 and 21 days' incubation, a small amount of the culture is mixed on a spot plate with phenol red indicator. An alkaline reaction signifies use. Paenibacillus l. larvae does not use citrate.
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| | | Growth in nutrient broth (11): bacteria are inoculated into a tube of nutrient broth (3 g beef extract, 5 g peptone, 1000 ml distilled water) and incubated either until growth occurs or for 14 days. If the culture grows, a loopful is transferred to another tube of nutrient broth. This procedure is repeated serially either for ten successive serial transfers or until growth fails. Only cultures that survive ten serial transfers are considered able to grow in nutrient broth. Paenibacillus l. larvae is unable to withstand serial transfer in nutrient broth (1). On the contrary, P. l. pulvifaciens can grow on this routine medium (15).
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| | | Liquefaction of gelatin (11): cultures in tubes of plain gelatin (120 g gelatin, 1000 ml distilled water, pH 7.0) incubated at 28°C are tested for liquefaction at 3- to 4-day intervals for 4 weeks. Before testing, cultures are placed at 20°C for approximately 4 hours to allow the unchanged gelatin to harden. Paenibacillus l. larvae causes gelatin to liquefy (1).
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| | | The use of commercial kits, such as API 50 CHB (5) and BBL CRYSTAL (8), for the biochemical characterisation of P. l. larvae can be taken into consideration. However, as it was proven that these kits produce different results for some of the biochemical reactions, a profile for P. l. larvae has to be drawn up for each system independently (8).
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| | e) | Polymerase chain reaction
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| | | The PCR is a genetic fingerprint technique that permits the identification of suspicious bacterial isolates and the detection of P. l. larvae in clinically and subclinically diseased larvae and honey bee products. Sample treatment depends on the application of the test.
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One colony of a suspicious colony is suspended in 50 µl of distilled water and heated to 95°C for 15 minutes (12). Following centrifugation at 5000 g for 5 minutes, 1 µl of the supernatant is used as template DNA in a PCR 50 µl mixture containing 2 mM MgCl2, 50 pmol of a forward and a reverse primer (primer sequences are given below), a 25-200 mM concentration of each deoxynucleoside triphosphate, and 1-1.25 U of Taq polymerase. Amplification of a specific DNA fragment occurs in a thermocycler under to the following PCR conditions: a 95°C step (1-15 minutes); 30 cycles of 93°C (1 minute), 55°C (30 seconds) and 72°C (1 minute); and a final cycle of 72°C (5 minutes). The molecular weights of the PCR products are determined by electrophoresis in a 0.8% agarose gel and staining with ethidium bromide.
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| | | The remains of two diseased honey bee larvae are suspended in 1 ml of sterile distilled water and mixed thoroughly; 100 µl of this suspension is diluted with 900 µl distilled water. This dilution is vortexed and 100 µl of it is used to extract DNA by heating and centrifugation (see above) (9). The PCR method remains the same for the different applications.
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The above-mentioned preparation method for template DNA based on heating and centrifugation can only be used when vegetative stages of the bacteria are present. The extraction of DNA from spores demands another approach. Indeed, spore suspensions are centrifuged at 6000 g and 4°C for 30 minutes. The pellet is then subjected to microwave treatment for 5 minutes at maximum power to break the spores, and the released DNA is suspended in 30 µl of 10 mM Tris/HCl, pH 8.0, containing 1 mM ethylene diamine tetra-acetic acid (EDTA). When spores are to be detected from honey, DNA was serially diluted with sterile distilled water to eliminate PCR inhibition caused by honey (28). Another DNA extraction method, based on lysozyme and proteinase K treatment, has been described (4).
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| | | Good results can also be obtained by incubating a pelleted spore suspension (for instance, from a honey sample or subclinically infected larvae) in MYPGP broth at 37°C for 2-24 hours. Thereafter, the suspension is centrifuged at 14,500 g for 5 minutes, washed with sterile distilled water and resuspended in 200 µl of sterile distilled water. This short incubation step causes spores to hatch, making them sensitive for DNA preparation by heat treatment again (see above) (22).
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| | | Several primer combinations based on the 16S rRNA gene are proven to be species specific. Differentiation between P. l. larvae and P. l. pulvifaciens is possible with the primer set provided by Piccini et al., when the number of cycles is reduced from 30 to 25 (28). The sequences of the primers are:
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Ref.
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Direction
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Sequence
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PCR-product size
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Specificity level
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| (12) |
forward
reverse |
5'-AAG-TCG-AGC-GGA-CCT-TGT-GTT-TC-3'
5'-TCT-ATC-TCA-AAA-CCG-GTC-AGA-GG-3' |
973 bp |
species |
| (9) |
forward
reverse |
5'-CTT-GTG-TTT-CTT-TCG-GGA-GAC-GCC-A-3'
5'-TCT-TAG-AGT-GCC-CAC-CTC-TGC-G-3' |
1106 bp |
species |
| (28) |
forward
reverse |
5'-CGA-GCG-GAC-CTT-GTG-TTT-CC-3'
5'-TCA-GTT-ATA-GGC-CAG-AAA-GC-3' |
700 bp |
subspecies |
| | | Identification of the two subspecies is also possible by further digest of a PCR-amplified 16S rDNA fragment with the endonuclease HaeIII (3). For the latter, the primers to be used have a much lower specificity and amplify the 16S rRNA genes from Bacillus, Paenibacillus, Brevibacillus and Virgibacillus species.
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| | f) | Modified hanging-drop technique
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| | | The modified hanging-drop technique (21) can be done directly on larval remains. However, due to its low specificity this technique is not conclusive.
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| | | Suspect material is mixed with water, and a drop of this suspension is placed on a cover-slip, dried and fixed by heat, and stained with carbol fuchsin or a suitable spore stain for 30 seconds. Any excess stain is washed off with water. While the preparation is still wet, the cover-slip is inverted on to a slide on which there is a very thin layer of immersion oil. Excess water will emerge. The slide is gently blotted dry and examined by high-power microscopy. By examining fields where pockets of water have formed in the oil, the spores of P. l. larvae will be seen exhibiting Brownian movement. Spores from other bacilli often remain fixed; in addition this technique allows the microscopic examination of the characteristic morphology of the foulbrood spores. If the infection is under 10 days old, long vegetative forms of the bacterium are present and some newly formed spores may be seen (24).
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| | g) | The Holst milk test
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| | | The Holst Milk test (17) uses the fact that a high level of proteolytic enzymes is produced by sporulating P. l. larvae. The test is performed by suspending a suspect scale, or a smear of a diseased larva, in a tube containing 1-4 ml of 1% powdered skim-milk in water. The tube is then incubated at 37°C. If P. l. larvae is present, the suspension will clear in 10-20 minutes.
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| | h) | Antibody-based techniques
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| | | Different antibody-based techniques have been developed for the diagnosis of American foulbrood. Most of them rely on polyclonal rabbit serum developed against pure cultures of P. l. larvae. In an immunodiffusion test, the antibodies interact with the bacterial antigen during a double diffusion process, leaving precipitation marks behind (27). In the fluorescent antibody technique these antibodies are conjugated with a fluorochrome dye. The resulting fluorescent antibody reacts with a bacterial smear on a slide. Any excess antiserum is washed off and the smear is examined by fluorescence microscopy. Paenibacillus l. larvae stains specifically as brightly fluorescing bacteria on a dark background (26, 30, 33). Antibody-based techniques are useful when no cross-reactivity with other bacilli has been demonstrated, for instance against Paenibacillus alvei, often found in late phase European foulbrood.
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| | | An enzyme-linked immunosorbent assay using a monoclonal antibody specific to P. l. larvae exists (25).
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| 2. | Serological tests
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| | No serological tests are available.
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C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
No vaccines or diagnostic biological products are available.
ACKNOWLEDGEMENT
Illustrations by Karl Weiss, extracted from Bienen-Pathologie, 1984, are reproduced with the kind permission of the author and Ehrenwirth-Verlag, Munich (Germany). Photographs are from the Central Science Laboratory, York (UK) and the Informatiecentrum voor Bijenteelt, Gent (Belgium) and published with kind permission of respecitively Ruth Waite and Frans J. Jacobs.
REFERENCES
| 1. | Alippi A.M. (1992). Characterization of Bacillus larvae White, the causative agent of American foulbrood of honey-bees. First record of its occurrence in Argentina. Rev. Argent. Microbiol., 24, 67-72.
|
| 2. | Alippi A.M. (1995). Detection of Bacillus larvae spores in Argentinean honeys by using a semi-selective medium. Microbiologica SEM, 11, 343-350.
|
| 3. | Alippi A.M., Lopez A.C. & Aguilar O.M. (2002). Differentiation of Paenibacillus larvae subsp. larvae, the cause of American foulbrood of honeybees, by using PCR and restriction fragment analysis of genes encoding 16S rRNA. Appl. Environ. Microbiol., 68 (7), 3655-3660.
|
| 4. | Bakonyi T., Derakhshifar I, Grabensteiner E., Nowotny N. (2003). Development and evaluation of PCR assays for the detection of Paenibacillus larvae in honey samples: comparison with isolation and biochemical characterization. Appl. Eviron. Microbiol., 69 (3), 1504-1510.
|
| 5. | Carpana E., Marocchi L. & Gelmini L. (1995). Evaluation of the API 50CHB system for the identification and biochemical characterization of Bacillus larvae. Apidologie, 26, 11-16.
|
| 6. | de Graaf D.C., Vandekerchove D., Dobbelaere W., Peeters J.E. & Jacobs F.J. (2001). Influence of the proximity of American foulbrood cases and apicultural management on the prevalence of Paenibacillus larvae spores in Belgian honey. Apidologie, 32, 587-599.
|
| 7. | Dingmann D.W. & Stahly D.P. (1983). Medium promoting sporulation of Bacillus larvae and metabolism of medium components. Appl. Environ. Microbiol., 46, 860-869.
|
| 8. | Dobbelaere W., de Graaf D.C., Peeters J.E & Jacobs F.J. (2001). Comparison of two commercial kits for the biochemical characterization of Paenibacillus larvae larvae in the diagnosis of AFB. J. Apic. Res., 40, 37-40.
|
| 9. | Dobbelaere W., de Graaf D.C., Peeters J.E & Jacobs F.J. (2001). Development of a fast and reliable diagnostic method for American foulbrood disease (Paenibacillus larvae subsp. larvae) using a 16S rRNA gene based PCR. Apidologie, 32, 363-370.
|
| 10. | Gochnauer T.A. & Corner J. (1987). Detection and identification of Bacillus larvae in a commercial pollen sample. J. Apic. Res., 13, 264-267.
|
| 11. | Gordon R.E., Haynes W.C. & Pang C.H.N. (1973). The genus Bacillus. Agriculture Handbook No. 427, United States Department of Agriculture, Washington DC, USA.
|
| 12. | Govan V.A., Allsopp M.H. & Davison S. (1999). A PCR detection method for rapid identification of Paenibacillus larvae. Appl. Environ. Microbiol., 65, 2243-2245.
|
| 13. | Hansen H. & Brodsgaard C.J. (1999). American foulbrood: a review of its biology, diagnosis and control. Bee World, 80 (1), 5-23.
|
| 14. | Haynes W.C. (1972). The catalase test. An aid in the identification of Bacillus larvae. Am. Bee J., 112, 130-131.
|
| 15. | Heyndrickx M., Vandemeulebroecke K., Hoste B., Janssen P., Kersters K., De Vos P., Logan N.A., Ali N. & Berkeley R.C. (1996). Reclassification of Paenibacillus (formerly Bacillus) pulvifaciens (Nakamura 1984) Ash et al. 1994, a later subjective synonym of Paenibacillus (formerly Bacillus) larvae (White 1906) Ash et al. 1994, as a subspecies of P. larvae, with emended descriptions of P. larvae as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens. Int. J. Syst. Bacteriol., 46, 270-279.
|
| 16. | Hitchcock J.D. & Wilson W.T. (1973). Pathogenicity to honey bees of a strain of Bacillus larvae that does not reduce nitrate. J. Econ. Entomol., 66, 901-902.
|
| 17. | Holst E.C. (1946). A single field test for American foulbrood. Am. Bee J., 86, 14-34.
|
| 18. | Hornitzky M.A.Z. & Clark S. (1991). Culture of Bacillus larvae from bulk honey samples for the detection of American foulbrood. J. Apic. Res., 30 (1), 13-16.
|
| 19. | 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. J. Apic. Res., 32 (1), 51-52.
|
| 20. | Jelinski M. (1985). Some biochemical properties of Bacillus larvae White. Apidologie, 16 (1), 69-76.
|
| 21. | Kostecki R. (1969). Studies on improvement of control of American foulbrood of the honey bee (in Polish). Pszczelnicze Zeszyty Naukowe, 13, 97-135.
|
| 22. | Lauro F.M., Favaretto M., Covolo L., Rassu M. & Bertoloni G. (2003). Rapid detection of Paenibacillus larvae from honey and hive samples with a novel nested PCR protocol. Int. J. Food Microbiol., 81, 195-201.
|
| 23. | Lochhead A.G. (1937). The nitrate reduction test and its significance in the detection of Bacillus larvae. Can. J. Res., C15, 79-86.
|
| 24. | Michael A.S. (1957). Droplet method for observation of living unstained bacteria. J. Bacteriol., 74, 831-832.
|
| 25. | Olsen P.E., Grant G.A., Nelson D.L. & Rice W.A. (1990). Detection of American foulbrood disease of the honeybee, using a monoclonal antibody specific to Bacillus larvae in an enzyme-linked immunosorbent assay. Can. J. Microbiol., 36, 732-735.
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| 26. | Otte E. (1973). Contribution to the laboratory diagnosis of American foulbrood of the honey bee with particular reference to the fluorescent antibody technique. Apidologie, 4 (4), 331-339.
|
| 27. | Peng Y.S. & Peng K.Y. (1979). A study on the possible utilization of immunodiffusion and immunofluorescence techniques as diagnostic methods for American foulbrood of honeybees (Apis mellifera). J. Invertebr. Pathol., 33, 284-289.
|
| 28. | Piccini C., D'Alessandro B., Antunez K. & Zunino P. (2002). Detection of Paenibacillus larvae subsp. larvae spores in naturally infected bee larvae and artificially contaminated honey by PCR. World J. Microbiol. Biotechnol., 18, 761-765.
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| 29. | Shimanuki H. & Knox D.A. (1988). Improved method for the detection of Bacillus larvae spores in honey. Am. Bee J., 128, 353-354.
|
| 30. | Toshkov A., Valarianov T. & Tomov A. (1970). The immunofluorescence method and the quick and specific diagnosis of American foulbrood of beebrood (in German). Bull. Apic., 13, 13-18.
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| 31. | von der Ohe W. & Dustmann J.H. (1997). Efficient prophylactic measures against American foulbrood by bacteriological analysis of honey for spore contamination. Am. Bee J., 137 (8), 603-606.
|
| 32. | Woodrow A.W. (1941). Susceptibility of honey bee larvae to American foulbrood. Gleanings Bee Cult., 69, 148-151.
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| 33. | Zhavnenko V.M. (1971). Indirect method of immunofluorescence in the diagnosis of foulbrood (American and European) (in Russian). Veterinariia, 8, 109-111.
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*
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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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