Introduction

Inspection for bee disease is an important part of beekeeping. Apiary inspectors and beekeepers must be able to recognize bee diseases and parasites and to differentiate the serious diseases from the less important ones. The purpose of this publication is to acquaint readers with laboratory techniques used to diagnose diseases and to detect and identify parasites, pests, and other abnormalities of the honey bee. We realize that different laboratory methods are used by others; where possible, those methods are described. However, emphasis is placed on the techniques used by the U.S. Department of Agriculture Bee Research Laboratory. Directions for submitting samples for diagnosis or subspecies identification are included in appendixes A and B.

Methods of Diagnosing Disease

Techniques of Microscopic Examination

Most bee diseases can be diagnosed by observing the associated micro-organisms with a light microscope. The following techniques are commonly used to prepare microscope slides for examination:

Modified Hanging Drop

The modified hanging drop technique (Michael 1957) can be very useful for differentiating diseases of the brood. Residue from a suspected cell is first mixed with water. Then a drop of this suspension (smear) is placed on a cover glass. The suspension used should always be dilute and only slightly turbid. The smear is dried and fixed under a heat lamp, or the smear can be air dried and heat fixed by passing it rapidly through a Bunsen burner flame two or three times. The fixed smear is stained with carbol fuchsin² or a suitable spore stain for 10 seconds. Enough stain should be placed on the cover glass to cover the entire smear. The excess

¹Shimanuki is a microbiologist and Knox an entomologist at Bee Research Laboratory. Agricultural Research Service. U.S. Department of Agriculture. Beltsville. MD 20705.
²Solution A: 0.3 g basic fuchsin (90% dye content), 10 mL ethyl alcohol (95%); solution B: 5 g phenol. 95 mL distilled water. Mix solutions A and B.

stain is then washed off with water. While the smear is still wet, the cover glass is inverted with the smear side down and placed onto a standard microscope slide previously coated with a very thin layer of immersion oil. The slide is gently blotted dry and examined with a microscope using the oil immersion objective. Results: Organisms that are not heat fixed are caught in areas where pockets of water have formed in the oil, and the organisms usually exhibit Brownian movement (see section on American foulbrood).

Simple Stain

This method relies solely on differentiating the bacteria by morphology. Place a drop of the suspension directly on a microscope slide. Heat fix and stain the smear as described in the previous section. Carbol fuchsin, methylene blue, and safranin are examples of stains that can be used. Air-dry or gently blot the stained smear. Place a drop of immersion oil directly on the smear. No cover glass is necessary. Examine the slide, using the oil immersion objective. Results: Organisms are uniformly stained and easily distinguished.

Gram Stain

The Gram stain is a standard microbiological method that can be substituted for the simple stain. Briefly, the procedure is as follows: A fixed smear is stained with crystal violet, immersed in iodine solution, decolorized in ethyl alcohol, and counterstained with safranin. Results: Gram-positive organisms are blue; Gram-negative organisms are red.

Wet Mount

The wet mount is especially useful for examining fungi or protozoa. Macerate a portion of the sample in water. Place a drop of the suspension on a microscope slide, and carefully drop the cover glass on it to minimize air pockets. No stain is required. The wet mount is usually examined with the dry objectives of a microscope. Results: Organisms refract light and are therefore visible on the slide. A phase-contrast microscope may be helpful, especially if an oil immersion objective is required.

Microinjection Techniques

To diagnose some diseases or to determine toxic levels of sample materials, it may be necessary to feed or inoculate larvae, pupae, or adult honey bees. Michael (1960) developed a technique using a microinjector equipped with a syringe and a 30-gauge needle. The microinjector can be calibrated to repeatedly deliver uniform inoculating volumes as small as 1 µL. The apparatus can be used to introduce material orally into the midgut (ventriculus) of a larva or to feed individual adult honey bees. The microinjection technique can also be adapted to direct injections into the body cavity of larvae, pupae, and adults.

Collecting Honey Bee Larvae and Pupae

Honey bee larvae 3 to 5 days old are readily obtained by removing a brood frame containing the desired larvae from a colony and placing it horizontally above a towel-lined tray in an incubator at 34 degrees C. Within a few hours, the larvae crawl from their cells and drop to the tray below. Pupae can be easily obtained by collecting 5-day-old larvae as described above and incubating them in petri dishes until pupation occurs. This method of collecting larvae and pupae in large numbers saves considerable time and labor. It also eliminates the damage that can occur when attempting to remove these immature forms from cells by mechanical means.

Oral Introduction

Larvae

Honey bee larvae as young as 3 days and weighing as little as 25 mg can be force-fed by carefully inserting the needle through the mouthparts and into the esophagus. When the actuating lever is depressed, a predetermined volume of material is propelled through the esophagus and into the midgut, with no physical damage to the larva. After feeding, the larvae are placed in petri dishes lined with filter paper and incubated at 34 degrees C.

Adults

Individual adult honey bees can also be fed known volumes using the microinjector. Adult bees are collected and held in a cage for about 4 hours without food. The material to be fed, at the final concentration, should be mixed into a sucrose solution to make it attractive to the bees. The microinjector is first actuated to produce a known volume of liquid at the tip of the needle. Then a bee from the cage is grasped by the wings, held up to the drop of liquid, and allowed to feed. Cold temperatures can be used to slow the bees and make them easier to handle. Avoid the use of carbon dioxide as an anesthetic to aid in handling bees, because they are reluctant to feed after exposure to carbon dioxide and their longevity is reduced. After feeding, the bees are placed in small cages with a supply of sugar syrup and held in an incubator at 34 degrees C.

Direct Injection

Care should be taken to insert only the tip of the needle into the hemocoel and to not exceed inoculating volumes of 2 µL per bee.

Brood

Injections are usually confined to 4- to 5-day larvae. The larva is held gently between the first and second fingers and the thumb, and the larva must be absolutely parallel to the needle. After the integument is punctured by the needle under gentle pressure, the inoculum is expelled directly into the dorsal blood vessel; the needle is then withdrawn in a slow, steady movement. Any excessive pressure on the larva by the fingers, particularly when withdrawing the needle, must be avoided to prevent bleeding. Pupae can be inoculated dorsally between the third and fourth abdominal segments or through the propodeum of the thorax. After injection, the larvae or pupae are placed in petri dishes lined with filter paper and incubated at 34 degrees C. If excessive bleeding has occurred, it can be seen on the filter paper.

Pupae can also be left in brood combs and inoculated in the head capsule (Wilson 1970). The pupal head is exposed by removing the cell cap, and the needle is inserted between the ocelli or through the clypeal sclerite.

Adults

Adult bees can be injected either through the propodeum of the thorax or dorsally through the intersegmental membrane between the third and fourth abdominal segments. Adult bees should be carefully subjected to carbon dioxide anesthetic before and during the process of injection. When bee longevity is a factor in the test, cold temperature can be used as an anesthetic. After injection, the bees are placed in small cages with a supply of sugar syrup and held in an incubator at 34 degrees C.

Removal of Digestive Tract

Intact digestive tracts that have been removed from adult honey bees are very useful for the detection of protozoan diseases. The digestive tracts can be easily obtained by removing the head of the bee to free the digestive tract, grasping as much of the stinger as possible with a pair of fine tweezers, and then with a steady, gentle pull withdrawing the entire digestive tract. Freshly killed honey bees are required for this procedure.

Brood diseases

Brood combs from healthy colonies typically have a solid and compact brood pattern. Almost every cell from the center of the comb outward contains an egg, larva, or pupa. The cappings are uniform in color and are convex (higher in center than at margins). The unfinished cappings of healthy brood may appear to have punctures, but since cells are always capped from the outer edges to the middle, the holes are always centered and have smooth edges. In contrast, brood combs from diseased colonies usually have a spotty brood pattern (pepperbox appearance), and the cappings tend to be darker, concave (sunken), and punctured. Also, the combs may contain the dried remains of larvae or pupae (scales), which are found lying lengthwise on the bottom side of brood cells. Sometimes scales are difficult to locate because of the condition of the comb. Scale material can be easily located using long-wave ultraviolet or nearultraviolet light. Exposure to wavelengths of 3100-4000 angstroms will cause scale material to fluoresce. Some discretion must be used with this technique because honey and pollen may also fluoresce. Symptoms of various brood diseases are summarized in table 1.

Symptoms of a contagious disease are sometimes mimicked because of an unrelated factor. For instance, often brood that is neglected because of a shortage of nurse bees will die from either chilling or starvation.
Table 1 Comparative symptoms of various brood diseases of honey bees

Symptom	American Foulbrood	European Foulbrood	Sacbrood	Chalk brood
Appearance of brood comb	Sealed brood. Discolored, sunken, or punctured cappings.	Unsealed brood. Some sealed brood in advanced cases with discolored, sunken or punctured cappings.	Sealed brood. Scattered cells with punctured cappings. often with two holes	Sealed and unsealed brood.
Age of dead brood	Usually older sealed larvae or young pupae.* Lying lengthwise in cells.	Usually young unsealed larvae; occasionally older sealed larvae. Typically in coiled stage.	Usually older sealed larvae. Length wise in cells.	Usually older larvae. Lengthwise in cells.
color of dead brood	Dull white, becoming light brown, coffee brown to dark brown, or almost black.	Dull white, becoming yellowish white to brown, dark brown, or almost black.	Grayish or straw colored becoming brown, grayish black, or black; head and end darker.	Chalk white. Sometimes mottled with black spots
Consistency of dead brood	Soft, becoming sticky to ropy.	Watery; rarely sticky or ropy. Granular.	Watery and granular; tough skin forms a sac.	Pasty
Odor of dead brood	Slightly to pronounced putrid odor.	Slightly to penetrating sour.	None to slightly sour.	Yeast like, nonobjectionable
Scale characteristics	Lies uniformly flat on lower side of cell. Adheres tightly to cell wall. Fine, threadlike tongue of dead maybe present. Head lies flat. Black in color.	Usually twisted in cell. Does not adhere to cell wall. Rubbery. Black in color.	Head prominently curled towards center towards center of cell. does not adhere tightly to cell wall. Rough texture. Brittle. Black, in color.	Mummified. does not adhere to cell walls. Brittle. Usually chalky white in color. Sometimes black fruiting bodies are present
*Bold text indicates the most useful field characteristic

Symptoms can also be the result of a failing queen, laying workers, toxic chemicals, or poisonous plants. (See section on Noninfectious Diseases.)

Bacterial Diseases

American Foulbrood

Bacillus larvae is the bacterium that causes American foulbrood disease (AFB). Bacillus larvae is a slender rod with slightly rounded ends and a tendency to grow in chains. The rod varies greatly in length, from about 2.5 to 5 microns (µm), and is about 0.5 µm wide. The spore is oval and approximately twice as long as wide, about 0.6 by 1.3 µm. When stained with carbol fuchsin, the spore walls appear reddish purple and quite clear in the center. The spores may form clusters and appear to be stacked. Approximately 2.5 billion spores are produced in each infected larva. If the larva has been infected for less than 10 days, the vegetative cells are present, and some newly formed spores may be seen.

The modified hanging drop technique can be very useful for differentiating American foulbrood from other brood diseases. In areas of the smear where pockets of water are formed in the oil, the spores of Bacillus larvae exhibit Brownian movement. This is an extremely valuable diagnostic technique because the spores formed by the other Bacillus species associated with the known bee diseases usually remain fixed. It is important to note that Brownian movement can be affected by slide preparation; also, debris and other bacteria can exhibit this motion. Therefore, Brownian movement must not be used as the sole criterion for diagnosis but must be considered together with the characteristic morphology of the spores and the gross larval symptoms. If microscopic examination is not conclusive, cultural tests can be made using the same suspension.

Cultivation of Bacillus larvae

Thiamine (vitamin B) and some amino acids are required for the growth of Bacillus larvae. Routine culture media such as nutrient broth will not support the growth of this organism. Good vegetative growth occurs on Difco brain heart infusion fortified with 0.1 mg thiamine hydrochloride per liter of medium (BHIT) and adjusted to pH 6.6 with HCl, but sporulation does not occur. Satisfactory growth and sporulation occur on the yeast extract, soluble starch, and glucose media recommended by Bailey and Lee (1962). The medium can be liquid, semisolid (0.3% agar), or solid (2% agar). For more information on sporulation, see Dingman and Stahly (1983).

Bacillus larvae spores also reproduce in the hemolymph of honey bee larvae, pupae, and adults when artificially introduced by injection (Michael 1960, Wilson and Rothenbuhler 1968, Wilson 1970).

To culture Bacillus larvae, we prepare spore suspensions by mixing diseased material (scales) with 9 mL sterile water in screw-cap tubes. (We use cotton swab applicators to remove and transfer the scales from


Table 2. Differentiation of Bacillus species in honey bees

Species	Brownian movement *	Catalase Nitrate production	nutrient reduction	Growth on agar
Bacillus larvae	+	-	+	-
Bacillus alvei	-	+	-	+
Bacillus laterosporus	-	+	+	+
Bacillus pulvifaciens	-	-	+	+

*In modified hanging drop technique.

the comb to the tubes.) The suspension is heat shocked at 80 degrees C for 10 minutes (effective time) to kill nonsporeforming bacteria. A sterile cotton swab is used to evenly spread a portion of the suspension (approximately 0.2 mL) over the surface of BHIT agar plates, which are then incubated for 72 hours at 34 degrees C. Individual colonies are small (1-2 mm) and opaque; however, if large numbers of viable B. larva spores are inoculated, a solid layer of growth will cover the plate.

There are no reliable methods for making plate counts of Bacillus larvae because fewer than 10 percent of the spores will produce visible growth on the media presently available (Shimanuki 1963). By calibrating our methods using spore and plate counts, we have determined that a minimum of 100 B. larvae spores are required to produce visible growth on BHIT.

Diagnostic Tests for Bacillus larvae

Holst Milk Test. The holst milk test (Holst 1946) is a simple test based on the fact that a high level of proteolytic enzymes is produced by sporulating Bacillus larvae. The test is conducted by suspending a suspect scale or a smear of a diseased larva in a tube containing 3-4 mL of 1% powdered skim milk in water. The tube is then incubated at 37 degrees C. If B. larvae is present, the suspension should clear in 10-20 minutes. It should be noted that this test is not always reliable.

Nitrate Reduction. Bacillus larvae reduces nitrate to nitrite (Lochhead 1937). The nitrate reduction test can be performed on a medium such as BHIT, which contains potassium nitrate (1-2 mg/ L of medium). After growth has occurred, the addition of a drop of sulfanilic acid-alphanaphthol reagent produces a red color if nitrate has been reduced to nitrite. Diagnosis should not be based on this test alone but on this test along with larval gross symptoms, bacterial morphology, and growth characteristics of the bacterial colony.

Catalase Production. A drop of 3% hydrogen peroxide is placed on an actively growing culture on a solid medium. Most aerobic bacteria break down the peroxide to water and oxygen and produce a bubbly foam, but Bacillus larvae is almost always negative for this reaction (Haynes 1972).

Fluorescent Antibody. The fluorescent antibody technique requires the preparation of specific antibodies stained with a fluorescent dye. Rabbits are injected with pure cultures of Bacillus larvae, and the active antiserum is collected and stained with a fluorescent dye. This fluorescent antiserum
is mixed with a bacterial smear on a slide and allowed to react. The excess antiserum is washed off the slide, and the slide is then examined with a fluorescence microscope. B. larvae appears as brightly fluorescing bodies on a dark background (Toschkov et al. 1970, Zhavnenko 1971, Otte 1973, Peng and Peng 1979).

Viability Test for Bacillus larvae

One method of controlling AFB is to destroy the viability of Bacillus larvae spores in contaminated bee equipment. This can be accomplished by gamma or electron beam irradiation or by fumigation with a sterilant gas such as ethylene oxide. Assessment of the efficacy of these methods should be based on the number of spores remaining viable in a test sample of brood comb containing at least 10 AFB scales.

A spore suspension is prepared from the sample comb by mixing 10 scales in 10 mL sterile water. Since each scale contains about 2.5 billion spores (Sturtevant 1932), 1 mL of the suspension should contain 2.5 billion spores. A portion of the suspension (0.2 mL = 500 million spores) is spread onto solid BHIT plates as previously described and incubated for 72-96 hours.

The results of the viability tests are reported as the approximate number of viable spores on a per-scale basis. If no colonies form on the medium, the results are recorded as <100 viable spores per scale; 1-9 colonies are recorded as <1,000 per scale; 10-99, <10~000 per scale; over 100 colonies per plate, >10,000 viable spores per scale; and when the plate is completely overgrown with colonies, the results are reported as no detectable reduction of viable spores.

Terramycin (Oxytetracycline) Resistance Tests

Isolates of Bacillus larvae can be screened for sensitivity to oxytetracycline based on the size of inhibition zones on agar plates. A spore suspension of the B. larvae isolate to be tested is spread on solid BHIT as described previously. A disk (BBL Sensi-Disc) containing 5 µg oxytetracycline is then placed on the plate, and the plate is incubated at 34 degrees C for 72 hours. The zones formed by sensitive strains usually average 50 mm in diameter. Alternatively, oxytetracycline incorporated into liquid BHIT will inhibit the growth of sensitive strains of B. larvae at concentrations as low as 12 µg/L of medium (Gochnauer 1953). Care is necessary in making these tests, and adequate control strains should be included.

Any substantial reduction of the zone size or an increase in the concentration of oxytetracycline required to prevent growth of Bacillus larvae in liquid medium would be evidence of the development of resistant strains (Gochnauer et al. 1975). However, when interpreting the results of the tests, the effects of growth rates should be considered. Because strains often grow at different rates, one may falsely conclude that a strain is resistant. No resistance of B. larvae to oxytetracycline has yet been reported.

Detection of Bacillus larvae Spores in Hive Products

Honey. Occasionally it may be necessary to examine honey for the presence of Bacillus larvae. Due to the high concentration of carbohydrate and other natural bacteriostatic substance(s) in honey, the examination of honey requires special considerations. The classical method (Sturtevant 1932, 1936) is to dilute the honey 1:9 with water, centrifuge the mixture to concentrate the spores in the sediment, and then examine the sediment microscopically for the presence of spores.

We (Shimanuki and Knox 1988) have developed the following technique to detect Bacillus larvae spores in honey. Honey to be examined is heated to 45 degrees C to permit easier handling and to decrease viscosity for more uniform distribution of any spores that may be present. Twenty-five milliliters of honey is placed in a 50-mL beaker and diluted with 10 mL of sterile water. The diluted honey is then transferred into a 1.75-inch (44-mm) dialysis tube. The open end is tied after the tube is filled, and the tube is submerged in running water for 18 hours or in a water bath with three to four water changes in that period. Following dialysis, the contents of the tube are centrifuged at about 2,000 g for 20 minutes. The supernatant is carefully removed with a pipet to leave approximately 1 mL of residue. This residue is resuspended in 9 mL of water in a screw-cap vial and heat shocked at 80 degrees C for 10 minutes to kill nonsporeforming bacteria. Next, 0.5 mL of the suspension is spread onto a plate of BHIT agar. The plate is incubated at 37 degrees C for 72 hours and examined for colonies of B. larvae. Difficulties can sometimes occur when honey samples contain other sporeforming bacteria that may completely cover the surface of the plate.

Since approximately 100 Bacillus larvae spores are required to produce visible growth on BHIT, this technique can demonstrate the presence of B. larvae spores in samples that contain a minimum of 80 spores per mL of undiluted honey (25 mL honey X 80 sporesl/mL = 2,000 spores/dialysis = 2,000 spores in 10 mL or 200 spores/mL; 0.5 mL = a 100-spore inoculum). Lower spore levels can possibly be detected by the use of larger honey samples or a second centrifugation to further concentrate the spores.

Pollen. Bacillus larvae spores can also be recovered from bee-collected pollen pellets by physically removing bits of AFB scale. A series of sieves of different sizes is helpful. If scales are not detected, one may pass a water-pollen suspension through No.2 filter paper, centrifuge the filtrate, and culture the pellet as described above (Gochnauer and Corner 1974).

Beeswax. We have had some success in recovering spores morphologically similar to those of Bacillus larvae by melting beeswax in boiling water, removing the beeswax cake after cooling, and centrifuging the water at 2,000 g for 20 minutes. The sediment is then examined microscopically for the presence of spores. Spores have also been recovered from contaminated beeswax by chloroform extraction (Kostecki 1969). However, in both cases, positive identification of the spores is not possible because the recovery techniques render the spores nonviable.

European Foulbrood

Melissococcus pluton (= Streptococcus pluton) is the bacterium that causes European foulbrood disease (EFB). Melissococcus pluton has been reclassified into the new genus Melissococcus (Bailey and Collins 1982a and b). However, Melissococcus pluton has still not been adequately described to be accepted for the current editions of "Bergey's Manual of Determinative Bacteriology."

Melissococcus pluton is generally observed early in the infection cycle before the appearance of the varied microflora associated with this disease. The M. pluton cell is short, nonsporeforming, and lancet shaped. The cell measures 0.5-0.7 by 1.0 µm and occurs singly, in pairs, or in chains.

Cultivation of Melissococcus plutan

Melissococcus pluton can be isolated on a medium developed by Bailey (1959). The medium consists of 1 g yeast extract (Difco), 1 g glucose, 1.35 g potassium dihydrogen phosphate (KH2PO4), 1 g soluble starch, 2 g agar, and distilled water to make 100 mL; the pH is adjusted to 6.6 with potassium hydroxide (KOH) and the mixture is autoclaved at 10 lb per square inch (116 degrees C) for 20 minutes. It has been found that the addition of cysteine (0.1 g per 100 mL) improves the multiplication of M. pluton (Bailey and Collins 1982b).

It is difficult to isolate Melissococcus pluton on artificial media because of its growth requirements and the competition from other bacteria. Also, once isolated, identification of M. pluton is difficult due to its pleomorphic nature in culture. Melissococcus pluton is best isolated when few if any other organisms are present. According to Bailey (1959), it is best to dry smears of diseased larval midguts on a slide. A water suspension of this material or a suspension prepared from larvae (apparently healthy, infected, or dead), cappings, etc., can be streaked on freshly prepared Bailey's agar medium. Or decimal dilutions of these suspensions can be inoculated into molten Bailey's agar medium (45 degrees C) and poured into plates (Bailey 1981). The plates are incubated anaerobically at 34 degrees C. The "Gas Pak" (BBL) Anaerobic System, including a disposable hydrogen and carbon dioxide generator, is used to obtain anaerobic conditions. Small white colonies of M. pluton should appear after 4 days.

Serodiagnosis

Pinnock and Featherstone (1984) developed an enzyme-linked immunosorbent assay (ELISA) for detecting Melissococcus pluton. Using this technique, they were able to demonstrate the presence of M. pluton even in apparently healthy honey bee colonies.

Organisms Associated With European Foulbrood

Some organisms do not cause European foulbrood, but they influence the odor and consistency of the dead brood and can be helpful in diagnosis. These secondary invaders include the following:

Bacillus alvei.The bacterium Bacillus alvei is frequently present in cases of European foulbrood disease (EFB). It is a rod 0.5-0.8 µm wide by 2.0-5.0µm long. Spores measure 0.8 by 1.8-2.2 µm. Like Bacillus larvae, the spores may be clumped and appear stacked. The sporangium may be observed attached to the spore. Typical strains of B.alvei spread vigorously on nutrient agar and may show "motile colonies"; free spores may lie side by side in long rows on the agar. The growth of this bacterium produces an unpleasant odor.

Bacillus laterosponis (= Bacillus orpheus). Rods of Bacillus laterosporus measure 0.5-0.8 µm by 2.0-5.0 µm, and the spores 1.0-1.3 by l.2-l.5µm (figs). An important diagnostic feature is the production of a canoe-shaped parasporal body that stains very heavily along one side and the two ends, and remains firmly adherent to the spore after lysis of the sporangium. The clear portion with the finely outlined wall is the spore. B. laterosporus grows moderately on nutrient agar, becoming dull and opaque, and spreads actively if the agar surface is moist. Growth on nutrient agar with 1% glucose added (glucose agar) is thicker and may become wrinkled.

Enterococcus faecalis (= Streptococcus faecalis = Streptococcus apis = Streptococcus liquefaciens).
Ovoid cells (elongated in the direction of chain) are 0.5-1.0 µm in diameter and are usually in pairs or short chains. This organism resembles Melissococcus pluton and may exhibit Brownian movement when the modified hanging drop technique is used. Growth occurs on nutrient agar usually within 1 day. Colonies are generally smaller than 2 mm; they are smooth and convex, with a well-defined border. When magnified, the colonies appear light brown and granular.

Bacterium eurydice (= Achromobacter eurydice). There is no standard description of Bacterium eurydice. White (1912) described this organism as a small, slender Gram-negative rod with slightly rounded ends, occurring singly or in pairs and measuring 0.5-1.4 µm long by 0.4-0.7 µm wide. According to White (1920), Bacterium eurydice is best isolated by plating the midgut contents of infected larvae on glucose agar and incubating at room temperature. Growth is slow and never luxuriant, and colonies are convex, smooth, and glistening. However, later researchers, who were unable to isolate B.eurydice as described by White, used the name Bacterium eurydice for a Gram-positive bacterium isolated from diseased larvae. Therefore, mention of the name in the literature causes confusion. This organism is not included in the current editions of Bergey's manual.

Bacillus apiarius. The bacterium Bacillus apiarius is rarely encountered and may or may not be legitimately associated with EFB. Rods are 0.6-0.8 µm in diameter and often less at the poles. Special diagnostic features include the ridged, thick, rectangular spore coat and the stainable remnants of the sporangium, which remain attached for a considerable time. Growth can occur on Sabouraud dextrose medium.

Powdery Scale

Bacillus pulvifaciens is the bacterium that causes powdery scale disease. Powdery scale disease is seldom reported, perhaps because the average beekeeper is unable to identify it. A useful diagnostic characteristic is the scale that results from the dead larva. The scale is light brown to yellow and extends from the base to the top of the cell. The scale is powdery; when handled, it crumbles into a dust.

Bacillus pulvifaciens vegetative cells measure 0.3-0.6 µm by 1.5-3.0 µm. The spores are 1.0 by 1.3-1.5 µm. The bacterium can be isolated on nutrient agar, but growth is heavier on glucose agar. When first isolated, the organism produces a reddish-brown pigment that can be lost by subculturing. Bacillus pulvifaciens closely resembles Bacillus larvae, but the spores do not exhibit Brownian movement in the modified hanging drop technique. Also, B. pulvijaciens is distinguished by its ability to grow at 20 degrees C and by its growth on nutrient agar.

Fungal Diseases

Chalkbrood

Ascosphaera apis is the fungus that causes chalkbrood disease. Ascosphaera apis is a heterothallic organism and develops a characteristic spore cyst when opposite thallic strains (+ and -) fuse. Spore cysts measure 47-140 µm in diameter. Spore balls endosed within the cyst are 9-19 µm in diameter, and individual spores are 3.0-4.0 µm by 1.4-2.0 µm.

Chalkbrood disease can be easily identified by its gross symptoms. An affected larva becomes overgrown by fluffy cottonlike mycelia and swells to the size of the cell.

If only one strain (+ or -) of mycelium is present, the larva dries into a hard, shrunken, white chalklike mummy- thus the name chalkbrood. When the + and - mycelia are present in a diseased larva, spore cysts can form, and the resulting mummies appear either mottled (black on white) or completely black. In heavily infected hives, mummies can be found at the hive entrances or on the bottom boards. Mummies can sometimes be detected in brood cells by tapping the comb against a solid surface. This easy removal of larval remains also differentiates chalkbrood from other brood diseases.

Ascosphaera apis grows luxuriantly on potato dextrose agar fortified with 4 g yeast extract/L. Growth and sporulation also occur on malt agar but less profusely and with no aerial hyphae; this facilitates subculturing and microscopic examination. Cultures have a characteristic fruity odor similar to that of fermenting peaches. The optimum temperature for growth is 30 degrees C.

Ascosphaera apis can be easily isolated from newly infected larvae or fresh mummies. These can be placed directly on the medium and incubated. New mycelial growth is usually visible within 24 hours. Small blocks of agar containing mycelia can be transferred to new plates to obtain pure cultures and isolates of the + and - strains. A. apis can be isolated from old mummies by placing them on water agar (agar with no added nutrients), incubating them, and transferring the new mycelial growth to a nutrient medium. Difficulties sometimes occur because A. apis may fail to grow or may be overgrown by other fungi, which can contaminate old mummies.

If only one strain (+ or -) is isolated, a fluffy cottonlike growth will eventually cover the plate. When both the + and - thalli are isolated, spore cysts form throughout the culture. The + and - thalli are morphologically identical. They can be distinguished by inoculating isolates on opposing sides of a plate. When opposite thalli grow together, a line of spore cysts forms at the juncture.

Stonebrood

Stonebrood is usually caused by Aspergillus flavus, occasionally A. fumigatus, and sometimes other Aspergillus species. These fungi are common soil inhabitants that are pathogenic to adult bees, other insects, mammals, and birds. The disease is difficult to identify in its early stages of infection. The fungus grows rapidly and forms a characteristic whitish-yellow collarlike ring near the head end of the infected larva. A wet mount prepared from the larva shows mycelia penetrating throughout the insect. After death, the infected larva becomes hardened and quite difficult to crush-hence the name stonebrood. Eventually, the fungus erupts from the integument of the insect and forms a false skin. At this stage, the larva may be covered with green powdery fungal spores. The spores of Aspergillus flavus are yellow green, and A.fumigatus spores are gray green. These spores can become so numerous that they fill the comb cells that contain the affected larvae. Stonebrood can usually be diagnosed from gross symptoms, but positive identification of the fungus requires its cultivation in the laboratory and subsequent examination of its conidial heads . Aspergillus spp. can be grown on potato dextrose or Sabouraud dextrose agars.

Viral Disease: Sacbrood

Morator aetatulas is the virus that causes sacbrood disease. It is the only common brood disease that is caused by a virus. Since sacbrood-diseased larvae are relatively free from bacteria, laboratory verification is usually based on gross symptoms and the absence of bacteria. Positive diagnosis requires the use of a special antiserum. Affected larvae change from pearly white to gray and finally black. Death occurs when the larvae are upright, just before pupation. Consequently, affected larvae are usually found in capped cells. Head development of diseased larvae is typically retarded. The head region is usually darker than the rest of the body and may lean toward the center of the cell. When affected larvae are carefully removed from their cells, they appear to be a sac filled with water. Typically the scales are brittle but easy to remove. Sacbrood-diseased larvae have no characteristic odor.

Mixed Infections

Bacillius larvae produces a potent antibiotic that eliminates competition from other bacteria typically associated with honey bee larvae. For this reason, American foulbrood and European foulbrood are rarely found in the same colony, except in cases where AFB is just becoming established in colonies that already have EFB.

It is not unusual to find chalkbrood and sacbrood on the same comb or on a comb with larvae infected with AFB. However, no single larva has been found to be infected with more than one disease. This is an important point to remember when selecting a sample for disease diagnosis.