Diagnosis of

Honey Bee

Diseases United States

Department of

Agriculture

Agricultural

Research

Service

Agriculture

Handbook

Number 690
Diagnosis of

Honey Bee

Diseases

United States

Department of

Agriculture

Agricultural

Research

Service

Agriculture

Handbook

Number 690

Hachiro Shimanuki and David A. Knox
Abstract

Shimanuki, Hachiro, and David A. Knox. 2000. Diagnosis of Honey Bee

Diseases. U.S. Department of Agriculture, Agriculture Handbook No. AH–

690, 61 pp.

Apiary inspectors and beekeepers must be able to recognize bee diseases

and parasites and to differentiate the serious diseases from the less

important ones. This handbook describes laboratory techniques used to

diagnose diseases and other abnormalities of the honey bee and to identify

parasites and pests. Emphasis is placed on the techniques used by the U.S.

Department of Agriculture Bee Research Laboratory. Included are

directions for submitting, through APHIS-PPQ or state regulators, samples

of suspected Africanized honey bees for identification of subspecies. Also

included are directions for sending diseased brood and adult honey bees

for diagnosis of bee disease.

Keywords: Africanized honey bee, honey bee disease, honey bee disorder,

honey bee parasite, honey bee pest

Mention of trade names, commercial products, or companies in this

publication is solely for the purpose of providing specific information and

does not imply recommendation or endorsement by the U.S. Department

of Agriculture over others not mentioned.

While supplies last, single copies of this publication may be obtained at no

cost from Bee Research Laboratory, Building 476, BARC-East, Beltsville,

MD 20705.

Copies of this publication may be purchased from the National Technical

Information Service, 5285 Port Royal Road, Springfield, VA 22161;

telephone (703) 605–6000.

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DC 20250-9410, or call (202) 720–5964 (voice and TDD). USDA is an

equal opportunity provider and employer.

Issued April 1991; revised July 2000
Contents

Introduction .............................................................................................. 1

Symptoms ................................................................................................. 1

Bacterial Diseases ..................................................................................... 2

American Foulbrood ............................................................................ 2

European Foulbrood ............................................................................ 9

Powdery Scale ................................................................................... 12

Septicemia.......................................................................................... 13

Spiroplasmosis ................................................................................... 13

Fungal Diseases ...................................................................................... 14

Chalkbrood ........................................................................................ 14

Stonebrood ......................................................................................... 15

Protozoan Diseases ................................................................................. 16

Nosema Disease ................................................................................. 16

Amoeba Disease ................................................................................ 19

Other Protozoa ................................................................................... 19

Viral Diseases ......................................................................................... 20

Sacbrood ............................................................................................ 20

Chronic Bee Paralysis ........................................................................ 21

Filamentous Virus .............................................................................. 21

Acute Paralysis Bee Virus and Kashmir Bee Virus ........................... 22

Disease Interactions ................................................................................ 22

Parasitic Honey Bee Mites ..................................................................... 22

Varroa jacobsoni ................................................................................ 22

Honey Bee Tracheal Mite (Acarapis woodi) ..................................... 28

Tropilaelaps clareae .......................................................................... 33

Pests ........................................................................................................ 34

Wax Moths ......................................................................................... 34

Small Hive Beetle .............................................................................. 34

Bee-louse ........................................................................................... 37

Noninfectious Disorders ......................................................................... 37

Neglected Brood ................................................................................ 37

Overheating........................................................................................ 38

Genetic Lethality ............................................................................... 38

Plant Poisoning .................................................................................. 39

Pesticide Poisoning ............................................................................ 39

iii
Appendix A: Microscopy ....................................................................... 42

Appendix B: Microinjection Techniques ................................................ 45

Appendix C: Directions for Sending Diseased Brood

and Adult Honey Bees for Diagnosis to the

ARS Bee Research Lab .................................................................. 48

Appendix D: Identification of Africanized Honey Bees ........................ 50

Appendix E: Reclassification of Bacillus spp. Associated

With Honey Bees ........................................................................... 51

References .............................................................................................. 52

Tables

Table 1. Comparative symptoms of various brood diseases

of honey bees .............................................................................. 2

Table 2. Differentiation of spore-forming

bacteria in honey bees ................................................................ 4

Table 3. Comparative symptoms in honey bees poisoned

by toxic chemicals and selected plants ..................................... 40

Figures

Figure 1. Bacterium that causes American foulbrood disease .................3

Figure 2. Melissococcus pluton and Paenibacillus alvei .......................10

Figure 3. Bacteria associated with European foulbrood disease ........... 11

Figure 4. Spore cyst of Ascosphaera apis..............................................14

Figure 5. Conidial heads of Aspergillus flavus ......................................16

Figure 6. Nosema spores in a wet mount ...............................................16

Figure 7. Honey bee digestive tracts......................................................17

Figure 8. Cross sections of Malpighian tubules.....................................19

Figure 9. Life cycle of Varroa jacobsoni ...............................................24

Figure 10. Braula coeca ..........................................................................26

Figure 11. Varroa jacobsoni ....................................................................26

Figure 12. Morphological characters separating Acarapis species .........29

Figure 13. Honey bee trachea containing mites ......................................30

Figure 14. Positioning a bee for dissection .............................................31

Figure 15. Location of the trachea in the thorax .....................................31

Figure 16. Tropilaelaps clareae ...............................................................34

Figure 17. Greater wax moth, Galleria mellonella,

and lesser wax moth, Achroia grisella ...................................35

Figure 18. Small hive beetle, adults ........................................................36

Figure 19. Small hive beetle, larvae ........................................................36

Figure 20. Force-feeding a honey bee larva ............................................45

iv
1

Diagnosis of Honey Bee Diseases

Hachiro Shimanuki and David A. Knox

Introduction

Inspection for bee diseases 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 identify parasites, pests, and

other abnormalities of the honey bee and to acquaint readers with the

laboratory techniques used to diagnose diseases. We realize that different

laboratory methods are used by others; where possible, those methods are

described. However, we emphasize the techniques used at the Bee

Research Laboratory, of the Agricultural Research Service, U.S. Department of Agriculture.

Symptoms

Brood combs of 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 the center than at the 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.

By comparison, brood combs of diseased colonies usually have a spotty

brood pattern (pepperbox appearance), and the cappings tend to be darker,

concave (sunken), and punctured. The combs may contain the dried

remains of larvae or pupae (called scales), which are found lying lengthwise on the bottom sides of brood cells. Sometimes scales are difficult to

locate because of the condition of the comb. In such cases, scale material

can easily be located using longwave ultraviolet or near-ultraviolet light.

Exposure to wavelengths of 3,100 to 4,000 angstroms causes scale

material to fluoresce. Some discretion must be used with this technique

Shimanuki is a microbiologist and Knox an entomologist at the Bee Research

Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville,

MD 20705.
2

Table 1. Comparative symptoms of various brood diseases of honey bees

Appearance of Sealed brood. Unsealed brood. Sealed brood. Sealed and

brood comb Discolored, sunken, Some sealed brood Scattered cells unsealed brood.

or punctured in advanced cases with with punctured

cappings. discolored, sunken, cappings.

or punctured cappings.

Age of dead Usually older sealed Usually young unsealed Usually older Usually older

brood larvae or young pupae. larvae; occasionally sealed larvae; larvae. Upright

older sealed larvae. occasionally young in cells.

Typically in coiled unsealed larvae.

stage. Upright in cells.

Color of dead Dull white, becoming Dull white, becoming Grayish or straw- Chalk white.

brood light brown, coffee yellowish white to colored, becoming Sometimes

brown to dark brown, brown, dark brown, or brown, grayish mottledwith

or almost black. almost black. black, or black. black spots.

Head end darker.

Consistency of Soft, becoming sticky Watery; rarely sticky Watery and Watery to

dead brood to ropy. or ropy. Granular. granular; tough pastelike.

skin forms a sac.

Odor of dead Slight to pronounced Slightly sour to None to slightly Slight, nonbrood odor. penetratingly sour. sour. objectionable.

Scale Uniformly lies flat on Usually twisted in Head prominently Does not adhere

characteristics lower side of cell. cell. Does not adhere curled toward to cell wall.

Adheres tightly to cell tightly to cell wall. center of cell. Does Brittle. Chalky

wall. Fine, threadlike Rubbery. Black. not adhere tightly white, mottled,

tongue of dead pupae to cell wall. or even black.

maybe present. Head Rough texture.

lies flat. Brittle. Black. Brittle. Black.

Bold italics indicate the most useful field characteristics.

European foulbrood American foulbrood Sacbrood Chalkbrood Symptom

*

*
3

because honey and pollen

may also fluoresce. Symptoms of various brood

diseases are summarized in

table 1.

By contrast, the gross

symptoms of most adult bee

diseases are not unique. The

inability to fly, unhooked

wings, and dysentery, for

instance, are general symptoms associated with many

disorders. Symptoms of a

contagious disease are

sometimes mimicked because

of unrelated factors. For

example, a brood neglected

because of a shortage of nurse

bees will often die from either

chilling or starvation. Disease

symptoms can also be the

result of a failing queen,

laying workers, toxic chemicals, or poisonous plants (see

Noninfectious Disorders,

p. 37.).

Bacterial Diseases

American Foulbrood

Paenibacillus (formerly

Bacillus) larvae subsp.

larvae, hereafter referred to as

P. larvae, (see appendix E) is

the bacterium that causes

American foulbrood disease

(AFB). P. larvae is a slender

rod with slightly rounded

ends and a tendency to grow

in chains (fig. 1). The rod

varies greatly in length, from

Figure 1. Bacterium that causes

American foulbrood disease (not to

scale). Top, Paenibacillus larvae

vegetative cells. Middle, forming

P. larvae spores. Bottom, P. larvae

spores.
4

about 2.5 to 5 micrometers (m); it is about 0.5 m wide. The spore is

oval and about twice as long as wide, about 0.6 1.3 m. When stained

with carbol fuchsin, the spores have reddish-purple walls and clear

centers. They may form clusters and appear to be stacked. About 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. Adult bees are not susceptible to AFB.

The modified hanging drop technique (appendix A) 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, P. larvae spores

exhibit Brownian movement. This movement is an extremely valuable

diagnostic technique because the spores of other species associated with

known bee diseases usually remain fixed (see table 2). It is important to

note that Brownian movement can be affected by slide preparation, and

debris and other bacteria can exhibit this motion. Therefore, Brownian

movement must not be the sole diagnostic criterion but should be considered along 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 Paenibacillus larvae

Thiamine (vitamin B1) and some amino acids are required for the growth

of P. larvae. Routine culture media such as nutrient agar will not support

the growth of this organism. Good vegetative growth (but not sporulation)

occurs on Difco brain-heart infusion fortified with 0.1 mg thiamine

hydrochloride per liter of medium (BHIT) and adjusted to a pH of 6.6 with

Growth on

nutrient

agar

Nitrate

reduction

Catalase

production

Table 2. Differentiation of spore-forming bacteria in honey bees

Paenibacillus larvae +–+–

Paenibacillus alvei –+–+

Brevibacillus

 laterosporus – +++

Paenibacillus

 pulvifaciens ––++

 In modified hanging drop technique.

Species

Brownian

movement*

*
5

hydrochloric acid. 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 percent agar), or

solid (2 percent agar). P. 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).

For more information on sporulation, see Dingman and Stahly (1983).

To culture P. larvae, we prepare spore suspensions by mixing diseased

material (3 to 5 scales) with 9 ml sterile water in screw-cap tubes. (We use

moist cotton swabs to remove and transfer the scales from the comb to the

tubes.) The suspension is heat shocked at 80o

 C for 10 minutes (effective

time) to kill non-spore-forming bacteria. A sterile cotton swab or L-shaped

spreader is used to evenly spread about 0.2 ml of the suspension over the

surface of BHIT agar plates, which are then incubated for 72 hours at 34o

C. Individual colonies are small (1–2 mm) and opaque; however, if large

numbers of viable P. larvae spores are inoculated, a solid layer of growth

will cover the plate.

There are no reliable methods for making plate counts of P. larvae because

fewer than 10 percent of the spores produce visible growth on the presently available media (Shimanuki 1963). By calibrating our methods using

spore and plate counts, we determined that a minimum of 100 P. larvae

spores are required to produce visible growth on BHIT.

Diagnostic tests for Paenibacillus larvae

Holst milk test. The Holst milk test (Holst 1946) is a simple test based on

the high level of proteolytic enzymes produced by sporulating P. larvae.

The test is conducted by suspending a suspect scale or a smear of a

diseased larva in a tube containing 3 to 4 ml of 1-percent powdered skim

milk in water. The tube is then incubated at 37o C. If AFB is present, the

suspension should clear in 10 to 20 minutes. It should be noted that this

test is not always reliable.

Nitrate reduction. P. larvae reduces nitrate to nitrite (Lochhead 1937).

The nitrate reduction test can be performed on a medium such as BHIT,

with potassium nitrate added (1–2 mg/L of medium). After growth has

occurred, adding a drop of sulfanilic acid-alpha-naphthol reagent produces

a red color if nitrate has been reduced to nitrite. Diagnosis should not be

based on this test alone but, rather, on the test along with larval gross

symptoms, bacterial morphology, and growth characteristics of the

bacterial colony.
6

Catalase production. A drop of 3-percent 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 P. larvae is almost always negative for this reaction (Haynes 1972).

Fluorescent antibody. The fluorescent antibody technique requires

preparation of specific antibodies stained with a fluorescent dye. Rabbits

are injected with pure cultures of P. 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 (see appendix A). P. 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 Paenibacillus larvae

American foulbrood is transmitted by the spores of P. larvae. These spores

have been shown to remain viable for over 70 years (Shimanuki and Knox

1994). One method of controlling AFB is to destroy the viability of the

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 that remain viable in a test

sample of brood comb containing at least 10 AFB scales.

To test viability, prepare a spore suspension 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. Spread a portion of the suspension (0.2 ml = 500

million spores) onto solid BHIT plates as previously described (p. 5) and

incubate for 72 to 96 hours. The results of the viability test are reported as

the approximate number of viable spores per scale, as follows:

• 0 colonies form on the medium = <100 viable spores per scale

• 1–9 colonies = <1,000 viable spores per scale

• 10–99 colonies = <10,000 viable spores per scale

• over 100 colonies = >10,000 viable spores per scale

• plate is completely overgrown with colonies = no detectable reduction of viable spores.
7

Terramycin (Oxytetracycline hydrochloride)/Paenibacillus larvae

resistance tests

Isolates of P. larvae can be screened for sensitivity to oxytetracycline

hydrochloride (OTC) based on the size of inhibition zones on agar plates.

A spore suspension of the P. larvae isolate to be tested is spread on solid

BHIT as described previously. A disk (BBL Sensi-Disc) containing 5 g

tetracycline2

 is then placed on the plate, and the plate is incubated at 34o C

for 72 hours. The zones formed by sensitive strains usually are greater

than 50 mm in diameter. Alternatively, OTC incorporated into liquid BHIT

inhibits the growth of sensitive isolates of P. larvae at concentrations as

low as 12 g/L of medium (Gochnauer 1954). Care is necessary in doing

these tests, and adequate controls should be included.

Any substantial reduction of the zone size or an increase in the concentration of OTC required to prevent growth of P. larvae is evidence of the

development of resistance (Gochnauer 1954). However, when interpreting

test results, the effects of growth rates should be considered. Because

isolates of P. larvae grow at different rates, one may falsely conclude that

an isolate is resistant.

Detection of Paenibacillus larvae spores in hive products

Honey. Occasionally it may be necessary to examine honey for the

presence of P. larvae. Due to the high concentration of carbohydrate and

other natural bacteriostatic substance(s) in honey, the examination of

honey requires special considerations.

Dilution. 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 microscopically examine the sediment for the

presence of spores. However, conclusive demonstration of the presence of

P. larvae spores in honey requires cultural techniques. Hornitzky and

Clark (1991) report the following technique:

• Dilute a 125-ml honey sample with 250 ml phosphate-buffered saline

(pH 7.2) and centrifuge in 500-ml bottles for 45 minutes at 3,000 G.

• Pour the supernatant off, leaving approximately 3 ml of fluid, and

mix with the sediment in the bottle.

2 Tetracycline discs were substituted for oxytetracycline discs; the inhibition zones of

the tetracyclines are similar (Gochnauer 1970).
8

• Place a 0.5-ml aliquot in a 5-ml glass bottle and heat it in a water

bath at 80o C for 15 minutes. After heating, streak the suspension on

an agar plate containing an appropriate media.

• Incubate the agar plate at 37o C for 72 hours and examine it for

colonies of P. larvae.

Direct inoculation (Hansen 1984a,b). Place a 5-g aliquot of the honey in

50-ml sterile beakers and set in a water bath for 5 minutes (effective time)

at 88o

 to 92o C. After heating, consecutively streak three agar plates that

contain appropriate media, using an inoculating loop. Incubate the plates

at 37o C for 72 hours and examine for colonies of P. larvae. This method

can detect P. larvae when more than 2,000 spores are present in 1 g honey.

While quick and easy, the direct inoculation of honey onto agar plates is

limited by the amount of honey that can be sampled and the likelihood of

contaminants.

Dialysis (Shimanuki and Knox 1988). Heat the honey to 45o C to permit

easier handling and to decrease viscosity, allowing for more uniform

distribution of any spores. Place 25 ml of honey in a 50-ml beaker and

dilute with 10 ml of sterile water. The diluted honey is then transferred

into a 1.75-inch (44-mm) dialysis tube. Tie the open end after the tube is

filled, and submerge the tube in running water for 7 to 8 hours or in a

water bath, changing the water three or four times during the period.

Following dialysis, the contents of the tube are centrifuged at about 20,000

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 80o C for 10 minutes to

kill non-spore-forming bacteria. Next, spread 0.4 ml of the suspension

onto a plate of BHIT agar. Incubate the plate at 34o C for 72 hours and

examine for colonies of P. larvae.

Since about 100 P. larvae spores are required to produce visible growth on

BHIT, this technique can demonstrate the presence of P. larvae spores in

samples that contain a minimum of 80 spores per ml of undiluted honey

(25 ml honey 100 spores/ml = 2,500 spores/dialysis = 2,500 spores in

10 ml or 250 spores/ml; 0.4 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.

Difficulties can sometimes occur when honey samples contain Paenibacillus alvei or other spore-forming bacteria that may completely cover

the surface of the plate. Hornitzky and Clark (1991) overcame this problem by supplementing their media with 3 g/nalidixic acid/ml of media.
9

Pollen. Paenibacillus larvae spores can also be recovered from beecollected 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 in the dialysis method

(Gochnauer and Corner 1974).

Beeswax. We have had some success in recovering spores which are

morphologically similar to those of P. larvae by melting beeswax in

boiling water, removing the beeswax cake after cooling, and centrifuging

the water at 5,000 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

The bacterium Melissococcus pluton (= Streptococcus pluton) causes the

brood disease European foulbrood (EFB). Streptococcus pluton was

reclassified into the new genus Melissococcus by Bailey and Collins

(1982a,b). M. 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, non-spore-forming, and lancet shaped.

The cell measures 0.5–0.7 1.0 m and occurs singly, in pairs, or in

chains (fig. 2). When stained with carbol fuchsin, the organism appears

dark purple against a lighter background. Some distortion occurs during

the fixing and staining process; this can be reduced by negative staining.

M. pluton can also be detected using enzyme-linked immunosorbent

assays (Pinnock and Featherstone 1984), polyclonal antisera (Allen and

Ball 1993), or polymerase chain reaction (Govan et al. 1998).

Cultivation of Melissococcus pluton

M. 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 (KH2

PO4), 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 inch2 (116o C)

for 20 minutes. It has been found that the addition of cysteine (0.1 g/100

ml) improves the multiplication of M. pluton (Bailey and Collins 1982b).
10

It is difficult to isolate M. pluton on artificial media because of its growth

requirements and competition from other bacteria. Once isolated, identification of M. pluton is also difficult due to its pleomorphic nature. M.

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, and

so forth can be streaked on freshly prepared Bailey’s agar medium.

Decimal dilutions of these suspensions can also be inoculated into molten

Bailey’s agar medium (45° C) and poured into plates (Bailey and Ball

1991). The plates are incubated anaerobically at 34° C. [We use the “Gas

Pak” (BBL) Anaerobic System to obtain anaerobic conditions.] Small

white colonies of M. pluton should appear after 4 days. See Allen and Ball

(1993) for additional information.

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:

Paenibacillus alvei (=Bacillus alvei). The bacterium Paenibacillus alvei

is frequently present in cases of EFB. It is a rod 0.5–0.8 m wide 2.0–

Figure 2. Melissococcus pluton and Paenibacillus alvei (1,000).
11

5.0 m long (figs. 2 and 3).

Spores measure 0.8 1.8–2.2

m. Like P. larvae, P. alvei

spores may be clumped and

appear stacked. The sporangium

may be observed attached to the

spore. Typical cultures of P. alvei

spread vigorously on nutrient agar

and may show motile colonies.

The bacterium produces a sour

odor as it grows.

Brevibacillus laterosporus (=

Bacillus laterosporus = Bacillus

orpheus). Rods of Brevibacillus

laterosporus measure 0.5–0.8 

2.0–5.0 m, and the spores

measure 1.0–1.3 1.2–1.5 m

(fig. 3). 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 adhered 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-percent

glucose added (glucose agar) is

more luxuriant and may become

wrinkled.

Enterococcus faecalis (= Streptococcus faecalis = S. apis =

S. liquefaciens). Ovoid cells

(elongated in the direction of

chain) are 0.5–1.0 m in diameter

and are usually in pairs or short

chains (fig. 3). This organism

Figure 3. Bacteria associated with

European foulbrood disease (not to

scale). Top, Paenibacillus alvei.

Middle, Brevibacillus laterosporus.

Bottom, Enterococcus faecalis.
12

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 m long 0.4–0.7 m

wide. According to White (1920), Bact. 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 Bact. eurydice as described by White, used the name

Bact. eurydice for a gram-positive bacterium isolated from diseased

larvae. Therefore, mention of the name in the literature causes confusion.

Paenibacillus apiarius (= Bacillus apiarius). The bacterium

Paenibacillus apiarius is rarely encountered and may or may not be

legitimately associated with EFB. Rods are 0.6–0.8 m in diameter and

often smaller 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

Paenibacillus larvae subsp. pulvifaciens (= Bacillus pulvifaciens) (see

appendix E) is the bacterium suspected of causing powdery scale disease.

This disease is seldom reported because the incidence is low or, 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.

P. pulvifaciens vegetative cells measure 0.3–0.6 1.5–3.0 m. The spores

are 1.0 1.3–1.5 m. The bacterium can be isolated on nutrient agar, but

growth is more luxuriant on glucose agar. When first isolated, the organism produces a reddish-brown pigment that can be lost by subculturing.

P. pulvifaciens closely resembles P. larvae, but the spores do not exhibit

Brownian movement in the modified hanging drop technique. Also,
13

P. pulvifaciens is distinguished by its ability to grow at 20o C on nutrient

agar (see table 2).

Septicemia

Pseudomonas aeruginosa (= Pseudomonas apiseptica) is the bacterium

that causes septicemia in adult honey bees. This disease results in the

destruction of connective tissues of the thorax, legs, wings, and antennae.

Consequently, the affected bees fall apart when handled. Dead or dying

bees may have a putrid odor.

P. aeruginosa rods measure 0.5–0.8 1.5–3.0 m. They are gramnegative and occur singly, in pairs, or in short chains. A bacterial smear

and Gram stain (appendix A) can easily be prepared after removing a wing

from the thorax and dipping the wing base in a drop of water on a microscope slide. To isolate this organism, streak the base of a wing across

Difco Pseudomonas isolation agar or Pseudomonas agar F. The optimum

temperature for growth is 37o C. P. aeruginosa in culture is characterized

by the excretion of diffusible yellow-green pigments that fluoresce in

ultraviolet light (wavelength below 260 nanometers).

Septicemia can also be diagnosed by reproducing the disease symptoms in

healthy, caged bees. This is accomplished by preparing a water extract

(macerate the equivalent of one suspect bee per ml of water) and inoculating healthy bees in the thorax (see appendix B) or dipping them in the

water extract. Bees with septicemia die within 24 hours; they exhibit the

typical odor and “break apart” symptom after about 48 hours.

Spiroplasmosis

Spiroplasma species is the bacterium that causes spiroplasmosis in adult

honey bees. Spiroplasma is a helical, motile, cell-wall-free prokaryote that

is found in the hemolymph of infected adult honey bees. The organism is a

tiny, coiled, and sometimes branched filament 0.7–1.2 m in diameter. Its

length increases with age and ranges from 2 m to more than 10 m

(Clark 1977, 1978a).

Spiroplasma can best be seen in the hemolymph, using dark-field microscopy (appendix A). They can also be seen by using the oil-immersion

objective of a phase-contrast microscope. Hemolymph can be taken from

adult bees by puncturing the intersegmental membrane directly behind the

first coxae, using a fine capillary tube made from the tip of a Pasteur pipet.

This organism can be cultured in standard mycoplasma broth medium
14

(GIBCO) and in Singh’s mosquito tissue culture medium with 20-percent

fetal calf serum.

Fungal Diseases

Chalkbrood

The fungus Ascosphaera apis

causes chalkbrood disease. It is

a heterothallic organism and

develops a characteristic spore

cyst when opposite thalli (+ and

–) fuse (fig. 4). Spore cysts

measure 47–140 m in

diameter. Spore balls enclosed

within the cyst are 9–19 m in

diameter, and individual spores

are 3.0–4.0 1.4–2.0 m.

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 brood cell. If

only one thallic strain (+ or –) is present, the larva dries into a hard,

shrunken, white, chalklike mummy—thus the name chalkbrood. When the

+ and – thalli are present in a larva, spore cysts can form, and the resulting

mummies appear either mottled (black on white) or completely black. In

hives with large numbers of infected larvae, mummies can be found at the

hive entrances or on the bottom boards. Mummies can sometimes be

removed from 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 per liter. Growth and sporulation also occur on malt agar

but less profusely and with no aerial hyphae; these characteristics facilitate

subculturing and microscopic examination. Cultures have a characteristic

fruity odor similar to that of fermenting peaches. The optimum temperature for growth is 30o C.

Figure 4. Spore cyst of Ascosphaera apis

containing spore balls, which in turn

contain spores.
15

A. apis can be easily isolated from newly infected larvae or “soft” mummies. Place the larvae or mummies directly on the medium and incubate

them. 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 – thalli. A. apis can be

isolated from old mummies by placing the mummies 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 thallus (+ or –) is isolated, a fluffy cottonlike growth will

eventually cover the plate. When both the + and – thalli are present,

fruiting bodies 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 a larval disease usually caused by Aspergillus flavus.

Aspergillus fumigatus, A. niger, and sometimes other Aspergillus species

are associated with the disease. These fungi are common soil inhabitants

that are also 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 A. flavus are yellow

green, those of A. fumigatus are gray green, and those of A. niger, black.

These spores can become so numerous that they fill the comb cells

containing 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 (fig. 5). Aspergillus spp. can

be grown on potato dextrose or Sabouraud dextrose agars. Caution: These

fungi can cause respiratory diseases in humans and other animals.
16

Protozoan Diseases

Protozoa are predominately

microscopic and usually

occur as single cells. No

protozoa are commonly

found in association with the

brood of honey bees.

Nosema Disease

The protozoan Nosema apis

causes nosema disease in

adult honey bees. N. apis

spores are large, oval bodies,

4–6 m long 2–4 m wide

(fig. 6). The spores develop

exclusively within the

epithelial cells of the ventriculus of the adult honey

bee. The disease usually

manifests itself in bees that

are confined, so the heaviest

infections are found in winter

bees, package bees, bees

from hives used for

pollination in greenhouses,

and so on.

No single symptom

typifies the disease.

Differences between

healthy bees and heavily

infected bees can be seen

by removing the digestive

tract and examining the

ventriculus. The ventriculus of a healthy bee is

straw brown, and the

individual circular constrictions can be clearly

seen (fig. 7). In a heavily

Figure 5. Conidial heads of Aspergillus

flavus.

Figure 6. Nosema spores as they appear in a

wet mount (400).
17

infected bee, the ventriculus is white, soft, and swollen, and the constrictions are obscured (White 1918). However, positive diagnosis can be made

only by microscopic examination of suspect bees or their fecal material for

the presence of N. apis spores (appendix A). Samples of bees to be

examined can be dried or preserved in alcohol. In a partially decomposed

sample, the presence of yeasts and molds resembling N. apis may make

accurate diagnosis difficult.

For quick, routine examinations, the abdomens from 10 or more bees are

removed, placed in a dish with 1.0 ml water per bee abdomen, and ground

with a pestle or the rounded end of a clean test tube. A cleaner preparation

can be obtained by grinding digestive tracts removed from the abdomens.

A wet mount is prepared from the resulting suspension and examined

under the high dry objective of a compound microscope. Alternatively,

individual bees can be examined to obtain the approximate percentage of

infected bees in a colony.

Coprological examination

By examining fecal material, Nosema can also be detected without

sacrificing workers or queens. On glass plates collect feces of worker bees

near the hive entrance, scrape off a deposit, mix it with water, and prepare

a wet mount from the resulting suspension (Wilson and Ellis 1966).

Suspect queens can be confined in small petri dishes or in glass tubes and

allowed to walk freely. They usually defecate within 1 hour. Queen feces

appear as drops of clear, colorless liquid, which can then be transferred to

a microscope slide with a pipet or capillary tube. Place a cover glass over

the feces before examination with a high, dry objective (L’Arrivee and

Hrytsak 1964).

Figure 7. Honey

bee digestive

tracts. Top, from a

healthy honey

bee. Note the

individual circular

constrictions on

the ventriculus.

Bottom, from a

honey bee with

nosema disease.
18

Counting Nosema spores

A quantitative measure of levels of Nosema can be obtained using a

hemocytometer, an instrument used to count human blood cells. It consists

of a special cover glass and a ruled chamber that holds a specific volume

of fluid. These chambers cost about $100 and are available from scientific

supply houses. Remove a sample of the spore suspension made from the

abdomens, preferably with a loop, and place it under the cover glass.

When the droplet touches the chamber and cover glass, it will flow under

the cover glass, thereby filling the chamber and ensuring the correct

volume. Allow about 3 minutes for the spores to settle, but count them

before the sample begins to dry in the chamber.

To count, find the ruled area and focus the microscope on the spores so

they are sharply defined. The squares are arranged in groups of 16, with

each group bounded by double lines. Count all the spores in the block

bounded by the double lines. Score the spores that cross the double lines if

they are on the upper or right side but not if they are on the lower or left

side. A hand tally counter is convenient to count the cells within a square.

To obtain a good average, count five blocks of 16 small squares.

Since each small square in the chamber has dimensions of 0.05 0.05 

0.1 mm, the total volume is 0.00025 mm3

, that is, 1/4000 of 1 mm3. Find

the average number of spores per square and multiply this figure by 4,000

to obtain the number of spores per cubic millimeter. To determine the

number per cubic centimeter (milliliter), multiply the number per cubic

millimeter by 1,000. If you started with the equivalent of 1 ml of water for

each ground-up bee, you can use the following equation to determine the

number of spores per bee, which is equal to the number of spores per

cubic centimeter:

(total number of spores counted) (4 106

)/number of squares counted =

number of spores per bee.

The suspension must be diluted if the spores are so numerous that accurate

counts cannot be made. Usually, accurate counts can be obtained with a

dilution of 1 part spore suspension to either 1 or 9 parts water (which gives

a dilution factor of 2 or 10, respectively). This factor must then be

incorporated into the equation. For more detail see Cantwell (1970).
19

Removal of digestive tract

Intact digestive tracts that have

been removed from adult honey

bees are very useful for the

detection of protozoan diseases

(refer to fig. 7). The digestive tracts

can be obtained easily by removing

the bee’s head 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.

Amoeba Disease

Malpighamoeba mellificae causes

amoeba disease. Since this protozoan is found in the Malpighian

tubules of adult bees, diagnosis can

be made only by removing and

microscopically examining the

tubules for the presence of amoeba

cysts. The cysts measure 5–8 m in

diameter (fig. 8).

Malpighian tubules are long,

threadlike projections originating at

the junction of the midgut and the

hindgut. They can be teased away from the digestive tract with a pair of

fine tweezers. Once this is done, place them in a drop of water on a

microscope slide and position a cover glass over them, applying uniform

pressure to obtain a flat surface for microscopic examination. M.

mellificae can be discerned using a high, dry objective and then changing

to the oil immersion objective for more detail.

Other Protozoa

Gregarines

Four gregarines (protozoans of the order Gregarinida) are associated with

honey bees: Monoica apis, Apigregarina stammeri, Acuta rousseaui, and

Figure 8. Cross sections of Malpighian

tubules. Top, healthy tubule. Bottom,

tubule containing cysts of

Malpighamoeba mellificae.
20

Leidyana apis. The immature stages, or cephalonts, average about 16 44

m. Cephalonts are oval and consist of two distinct body segments; the

posterior segment is larger. The mature stages, or sporonts, average about

35 85 m and have a reduced anterior segment.

Gregarines are found attached to the epithelium of the midgut of adult

honey bees. To view gregarines, gently remove the midgut from the

digestive tract of a suspect bee and place it on a microscope slide in a drop

of water. The midgut can be separated from the digestive tract at the point

of attachment with the proventriculus (honey stomach) and hindgut, using

fine tweezers and a scalpel. Gently break open the midgut with fine

tweezers and a probe, and place a cover glass over the resulting suspension. Gregarines can be seen using the low-power objective of a compound microscope.

Flagellates

The flagellates associated with honey bees are Crithidia (= Leptomonas)

species. Flagellates have been found either free in the lumen or attached to

the epithelium of the hindgut and rectum of adult honey bees (Fyg 1954).

Flagellates vary in size from 5 to 30 m. Some appear as pearlike bodies

with flagella; others are long threadlike forms or are round without

flagellae (Lotmar 1946).

To look for flagellates, remove the digestive tract of a suspect bee as

described and place it in a drop of water on a microscope slide. Then,

using fine tweezers and a scalpel, separate the hindgut and rectum at the

point of attachment with the midgut. Macerate the hindgut and rectum,

using the tweezers and a probe. Place a cover glass on the resulting

suspension and observe under the high, dry objective of the microscope.

Viral Diseases

Sacbrood

Sacbrood is the only common brood disease 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 or

molecular techniques. 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
21

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.

Chronic Bee Paralysis

Adult bees affected by chronic bee paralysis are usually found on the top

bars of the combs. They appear to tremble uncontrollably and are unable

to fly. In severe cases, large numbers of bees are found crawling out the

hive entrance. Individual bees are frequently black, hairless, and shiny. In

some cases, paralysislike symptoms can be caused by toxic chemicals.

Ideally, the diagnosis of this disease is made using serological techniques.

Since this is beyond the capability of most laboratories, diagnosis is

usually made by observing symptoms in individual bees and, when

possible, colony behavior. Chronic paralysis can be diagnosed by reproducing the disease symptoms in caged bees. This can be done by spraying,

feeding, or injecting a water extract made from suspect bees. The extract is

prepared by macerating the equivalent of one suspect bee in 1 ml water.

This is then centrifuged to eliminate large suspended matter and passed

through a 0.45-m filter to remove bacteria. To feed up to 20 caged bees,

mix 2 ml of the extract with an equal volume of sugar syrup. For inoculation, each bee receives 1.0 l of the extract through a dorsal abdominal

intersegmental membrane (see appendix B). The symptoms of paralysis

should be visible after 6 days. Control bees should be treated with extracts

made from healthy bees.

Filamentous Virus

Filamentous virus is also known as F-virus and bee rickettsiosis. This

disease, previously thought to be of rickettsial origin, can be diagnosed by

examining the hemolymph of infected adult bees using dark-field or

phase-contrast microscopy. The hemolymph of infected honey bees is

milky white and contains many spherical to rod-shaped viral particles of a

size close to the limit of resolution for light microscopy. The viral particles

consist of a folded nucleocapsid within a viral envelope and are 0.4 0.1

m (Clark 1978b).
22

Acute Paralysis Bee Virus and Kashmir Bee Virus

Acute paralysis bee virus (APBV) and Kashmir bee virus (KBV) are two

serlogically related viruses, and the antiserum produced from one virus

will cross-react with the other virus (Hung et al. 1996). These viruses

commonly occur in apparently healthy adult bees. No specific gross

symptoms have been attributed to either virus. Whereas APBV is a disease

of adults, KBV is reported to cause mortality in brood and adult honey

bees. APBV and KBV diseases can be diagnosed using immunodiffusion

tests. Recently, molecular methods were developed for detecting both

diseases. However, immunodiffusion and molecular methods are not

routinely used in our laboratory.

Disease Interactions

Paenibacillus larvae produces a potent antibiotic that eliminates competition from other bacteria typically associated with honey bee larvae. For

this reason, American foulbrood (AFB) and European foulbrood (EFB) are

rarely found in the same colony, except in cases where AFB is just

becoming established in colonies that already have EFB.

Ascosphaera apis produces linoleic acid, which inhibits the growth of

Paenibacillus larvae and Melissococcus pluton. Since the introduction of

chalkbrood into the United States, the incidence of EFB has fallen dramatically. However, the incidence of AFB appears to have remained constant. It is not unusual to find chalkbrood and sacbrood on the same comb

or on a comb with larvae infected with AFB, although 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.

Mixed infections in adult bees are more common. Adult bees could be

infected with N. apis and also be infected with viruses or spiroplasma. It is

also possible for adult bees to be infested with one or more species of

mites.

Parasitic Honey Bee Mites

Varroa jacobsoni

The mite Varroa jacobsoni can be found on adult bees, on the brood, and

in hive debris. The adult female mite is oval and flat, about 1.1 mm long

1.5 mm wide, and pale to reddish brown; it can easily be seen with the

unaided eye. Because the mites attach to the adult bee between the
23

abdominal segments or between body regions (head, thorax, abdomen),

they are difficult to detect. However, they can be easily seen against the

white surface of pupae. Male mites are considerably smaller, are pale to

light tan, and are rarely encountered (Delfinado-Baker 1984).

The life cycle of V. jacobsoni is summarized in figure 9. It is important to

note that the bee-louse, Braula coeca, resembles V. jacobsoni in size and

color. However, being an insect, Braula has six legs that extend to the side

(fig. 10). Varroa, an arachnid, has eight legs that extend forward (fig. 11).

The most severe parasitism occurs on older bee larvae and pupae, with the

mite preferring drone brood to worker brood (Ritter and Ruttner 1980). In

heavy infestations, pupae may not develop into adult bees. Adults that do

emerge may have shortened abdomens, misshapen wings, and deformed

legs and may weigh less than healthy bees (De Jong et al. 1982b). The

misshaped wings are believed to be caused by deformed wing virus. V.

jacobsoni may act as a vector of the virus, introducing it to the pupae

during feeding.

Sampling techniques

When sampling, remember that the number and location of V. jacobsoni in

a colony vary according to time of year. The number of mites is lowest in

spring, increases during summer, and peaks in fall. During spring and

summer, most mites are found on the brood (especially drone brood). In

late fall and winter, most mites are attached to adult worker bees.

To examine adults, collect 200 to 300 bees. This can be done by brushing

the bees off the comb through a large-mouthed funnel (paper or cardboard,

for example) or directly into a container. Individual honey bees can be

examined with or without the aid of a hand lens or dissecting microscope.

When the mites are moving about on a bee, they are fairly easy to detect;

but once they attach themselves between abdominal segments, they may

be difficult to find. Mites can be collected from adult bees as follows:

Shaking. Varroa jacobsoni can be dislodged by shaking the bees in

liquids such as hot water, alcohol, or detergent solution. We recommend

70-percent alcohol (ethyl, methyl, or isopropyl); the alcohol kills the bees

and preserves them for use for other purposes, such as examination for

Acarapis woodi. De Jong et al. (1982a) found that hand-shaking bees in

alcohol for 1 minute dislodged about 90 percent of the mites. The mites

are collected by passing the bees and alcohol through a wire screen (8- to

12-mesh) to remove the bees and then sieving the alcohol through cotton

cloth. The cloth is then examined for mites.
24

1

Adult bee, with

Varroa feeding

on hemolymph

6

1-6 eggs developing

from egg to larva to

protonymph to

deutonymph

7

5-6 days

adult male

7-8 days

adult female

8

Mating within cell

9

Adult females leave cell

with emerging bee.

Male and immature stages

stay in cell.

10

Mite transfer through

close contact between bees

adult female

egg

protonymph

deutonymph

Figure 9. Life cycle of Varroa jacobsoni. (Courtesy of Roger A. Morse)
25

2

Mite enters cell with

larva of 5 to 5-1/2 days

3

Mite in bee food

4

Mite feeds on prepupa.

5

Female lays first egg 60 hours

after cell capping. Female

lays subsequent eggs at 30-

hour intervals.

Ether roll. This technique is a

rapid and efficient detection

method in the field and minimizes

the handling, shipping, and timeconsuming procedures associated

with shaking adult bees in alcohol

or other solvents that best require a

laboratory. Collect 200 to 300 bees

in a jar and anesthetize them with

ether delivered from an aerosol can

(this aerosol product is sold in

auto-parts stores as an aid to start

engines). A 1- to 2-second burst of

aerosol is adequate. Roll the jars

lengthwise for about 10 seconds.

The majority of mites dislodge

from their hosts and adhere to the

inside wall of the jar. To complete

the process, deposit the bee sample

on a white surface and spread it

around. This should cause any

remaining mites to fall onto the

white substrate. The bees should be

immediately emptied from the jar

because the mites tend to stick to

them if left in the jar for more than

a few minutes. The bees can be

washed (see shaking method

above) if a more precise count is

desired.

Powdered sugar method.

Reported to be more efficient than

the ether roll method, this method

does not require bees to be killed.

Use a wide-mouth, pint or quart

canning jar. Set aside the metal

ring from the two-piece lid; discard

the center portion. Cut a circle of

#8-mesh hardware cloth to fit

inside the ring. Collect 200 to 300
26

Figure 10.

Braula coeca,

dorsal view.

Figure 11.

Varroa

jacobsoni,

ventral view.

bees in the jar. Through the #8-mesh lid, add enough powdered sugar to

the jar to coat the bees, about 1 teaspoon to 1 tablespoon. Roll the jar

around to distribute the sugar, allow the jar to sit for a few minutes, then

invert it, and shake it over a piece of paper to recover the mites. The mites

and sugar will pass through the mesh to the paper. Pour the sugar and

mites into another jar fitted with a fine-mesh lid and shake, allowing the

sugar to escape. Then dump the mites on a clean sheet of paper and count

them. A brief shaking will usually recover 70 percent of the mites; if you

persist a little longer, 90 percent can be recovered (Ellis 2000).
27

Heating method. Live adult honey bees can be shaken into a wire-based

cage and placed in an oven over white paper. The bees are heated for 10 to

15 minutes at 46o or 47o C. The Varroa, if present, fall off the bees and can

be observed on the white paper (Crane 1978).

To look for mites on brood, examine the pupae (preferably drone) individually. V. jacobsoni can be easily seen against the white surface of

worker or drone pupae after they are removed from their cells. Remember

to also examine the bottom of the cells. It is suggested that a minimum of

100 drone pupae per colony be examined.

The classic method of pupal collection is to uncap each cell and remove

the pupa with forceps. Groups of pupae can be quickly and easily removed

by inserting a capping scratcher at an angle through the cappings and

lifting the brood and cappings upward (Szabo 1989). Alternatively, the

brood comb can be incubated at 37o C, followed by examination of all the

emerged bees and remaining brood.

Debris in a hive, such as wax particles, pollen, dead bees and brood, and

mites, normally falls to the hive floor and is removed by housecleaning

bees during warm weather. The collection and examination of hive debris

for mites can be facilitated by placing white construction paper on the hive

floor. Staple the paper under a wire frame constructed with a 1/4-inch

wood frame and 8- to 12-mesh wire and keeping a 1/4-inch separation

between paper and wire. The wire protects the paper and debris from the

bees. Examine the paper for mites, which can easily be seen against the

white background. A magnifying glass or dissecting microscope may be

helpful in locating the mites in the debris. Sticky boards or shelf paper

(with the adhesive surface exposed) help hold the mites. Substances such

as petroleum jelly, cooking sprays, oils, and tanglefoot have been used for

preparation of sticky boards.

The acaricides used to treat mite infestations can also be used in combination with the paper method to detect V. jacobsoni. Apistan is currently

approved and available for this purpose. After treatment, the mites drop to

the paper and are easily detected. It is important that the paper have a

sticky surface to hold any recovering mites.

A flotation method can be used to examine debris for V. jacobsoni. Place

hive debris in a jar or pan and cover with 98-percent alcohol. The mites

float to the surface while the heavier debris sinks (Ritter and Ruttner

1980).
28

Parasitic mite syndrome

Colonies infested with parasitic mites can display an array of symptoms

referred to as the parasitic mite syndrome. The syndrome affects both

adult bees and brood. It is quite likely this syndrome is similar to that

reported by Ball (1988) who refers to the condition as a “secondary

infection” in colonies infested with V. jacobsoni. Some of the symptoms

are listed below. Not all symptoms may be evident in a colony at a given

time:

1. Presence of V. jacobsoni

2. Reduction in adult bee population

3. Evacuation of hive by crawling adult bees

4. Queen supersedure

5. Spotty brood pattern

6. Affected brood can vary from C-stage larva to prepupa

7. Individual larva may appear

• twisted in the cell

• “molten” in the bottom of the cell

• light brown, gray to black in color

• watery to pasty consistency, resembling EFB, AFB, and

sacbrood

8. Scales are not brittle and are easy to remove

9. No typical odor

10. No characteristic microflora

Honey Bee Tracheal Mite (Acarapis woodi)

Three Acarapis species are associated with adult honey bees: Acarapis

woodi, A. externus, and A. dorsalis. Only A. woodi is known to be harmful.

All three species are difficult to detect and identify because of their small

size and similarity, so they are frequently identified by location on the bee

instead of by morphological characters. A. woodi can be diagnosed solely

on habitat; the position of other species on the host is a useful but not an

infallible characteristic. A. woodi lives exclusively in the prothoracic

tracheae. A. externus inhabits the membranous area between the posterior

region of the head and thorax or the ventral neck region and the posterior

tentorial pits. A. dorsalis is usually found in the dorsal groove between the

mesoscutum and mesocutellum and the wing bases. Morphological

characters differentiating these species are shown in figure 12. For

complete descriptions and illustrations see Delfinado-Baker and Baker

(1982).
29

The female A. woodi, or honey bee tracheal mite, is 143–174 m long and

the male 125–136 m. The body is oval, widest between the second and

third pair of legs, and whitish or pearly white with shining, smooth cuticle.

A few long hairs are present on the body and legs. This mite has an

elongate, beaklike gnathosoma with long, bladelike styles (mouthparts) for

feeding on the host.

Acarapis dorsalis:

Apodeme (A) full length

of propodosoma (P);

coxal plate (B) deep

indentation; tarsal joints

(C) <10m

Acarapis externus:

Apodeme (A) 2/3 length

of propodosoma (P);

coxal plate (B) variable

indentation; tarsal joints

(C) <10m

Acarapis woodi: Apodeme (A)

2/3 propodosoma (P); coxal

plate (B) shallow indentation;

tarsal joints (C) <10m

Figure 12. Morphological characters separating Acarapis species.
30

No one symptom characterizes this disease. An affected bee could have

disjointed wings and be unable to fly, have a distended abdomen, or both.

Absence of these symptoms does not necessarily indicate freedom from

mites. Positive diagnosis can be made only by microscopic examination of

the tracheae. A. woodi is the only mite found in the bee tracheae, so this is

an important diagnostic feature.

The trachea must be examined carefully for the presence of mites. The

trachea of a severely infested bee may have brown or black blotches with

crustlike lesions and be obstructed by many mites in different stages of

development (fig. 13). However, the trachea may not be discolored when

mites are present, and a cloudy or discolored trachea does not always

contain mites.

Sample collection and preservation

The population of A. woodi may vary seasonally. During the period of

highest bee population, the number of bees with mites is reduced. The

likelihood of detecting tracheal mites is highest in the fall and winter. In

sampling, one should try to collect either moribund bees that may be

crawling near the hive entrance or bees at the entrance as they leave or

return to the hive (older bees). These bees should be killed and preserved

Figure 13. Honey bee trachea containing mites (100).
31

in 70-percent ethyl, methyl,

or isopropyl alcohol as soon

as they are collected.

Methods for detecting A.

woodi

Classic technique. Pin the

bee on its back and remove

the head and first pair of

legs by pushing them off

with a scalpel or razor blade

in a downward and forward

motion (fig. 14). Using a

dissecting microscope,

remove the first ring of the

thorax (tergite of prothorax) with forceps. This exposes the tracheal trunks

in the mesothorax (fig. 15). When the infestation is light, it is necessary to

remove the trachea. Place the trachea in a drop of 85-percent lactic acid on

a glass slide, and cover with a cover glass for examination at 40to 100

on a compound

microscope.

Quick examination of a few bees.

Put the bee on its

back, grasp it with

forceps or

between your

thumb and

forefinger, and

remove the head

and first pair of

legs by pushing

them off with a

scalpel or razor blade in a downward and forward motion. Then with a

scalpel, razor blade, or fine pair of scissors, cut a thin transverse section

from the anterior face of the thorax in such a way as to obtain a disk. Place

the disk on a microscope slide and add a few drops of lactic acid. This

makes the material more transparent and helps to separate the muscle.

With the aid of a dissecting microscope, carefully separate the muscles

and examine the trachea. Suspicious trachea should be removed and

examined as in the classic technique above.

Scapel Needle

Esophagus Trachea

Salivary

glands

Tergite of

prothorax

Spiracle

Point of

“collar”

Wing

muscles

Figure 14. Positioning a bee for dissection.

Figure 15. Location of the trachea in the thorax.
32

Examination of large numbers of bees. Slice transverse-section thoracic

disks as described directly above, place them in a small dish containing 5-

to 10-percent potassium hydroxide (KOH), and incubate at about 37o C for

16 to 24 hours. The KOH dissolves the muscle and fat tissue, leaving the

trachea exposed. Examine the disk-trachea suspension under a dissecting

microscope. Suspect trachea should be removed and examined as in the

classic technique.

Examination of unpreserved, dry bees. Cut a few thoracic disks as

described in the quick method above, place them on a slide, and add a few

drops of 10-percent potassium hydroxide. Heat the slide gently for 1 to 2

minutes (do not boil), cover with a cover glass, crush the disks lightly, and

examine microscopically.

Methylene blue staining technique (Peng and Nasr 1985). Prepare

transverse-section disks from the thoraxes of 50 bees as described in the

quick method. Place the disks in a beaker of 8-percent potassium hydroxide solution, and heat to boiling while gently stirring continuously.

Remove the solution from the heat and continue stirring until the soft

tissues inside the disks are dissolved and cleared (about 10 minutes).

Excessive stirring and heating will damage the specimens and subsequently reduce the color intensity of the mites. Recover the disks from the

KOH by filtration through a perforated Tissue-Tek processing capsule.

After filtration, cover the processing capsule with a lid, place in a beaker,

and wash with tap water to remove the remaining KOH. Transfer the

processing capsule to a modified methylene blue staining solution (prepared by first dissolving 1-percent aqueous methylene blue and then

adding sodium chloride to make a 0.85-percent sodium chloride solution).

Immerse the capsule in that solution for 5 minutes and then in distilled

water for 2 to 5 minutes. Finally, rinse the capsule with 70-percent ethyl

alcohol. Examine the disks for stained mites within the tracheae under a

dissecting microscope.

Differentiation of live mites from dead mites (Eischen et al. 1986).

Anesthetize live bees with carbon dioxide and remove the abdomens with

a scalpel to prevent being stung during examination. Remove the head and

first pair of legs by holding each bee on its back and gently pushing this

section off with a downward and forward motion. Holding the bee in this

position, place each under a dissecting microscope and remove the first

ring of the thorax with fine forceps. This exposes the tracheal attachment

to the thoracic wall, which is often the only location of mites in a light
33

infestation. With tweezers, remove tracheae that appear abnormal and

transfer them to a glass slide; as the moist tracheae dry, they will adhere to

the slide. Then dissect the tracheae using a pair of fine needle probes.

Mites are considered dead if they do not move. Dead mites often appear

discolored and desiccated; living mites have a translucent gray or pearl

color and move when prodded with a probe. This is the method of choice

for evaluating chemicals used to control tracheal mites.

Serodiagnosis. Ragsdale and Furgala (1987) produced an antiserum

against extracts of Acarapis woodi-infested tracheae to be used as the

primary antibody in a direct enzyme-linked immunosorbent assay

(ELISA). Ragsdale and Kjer (1989) improved the ELISA technique,

making it reliable for the detection of A. woodi. Grant et al. (1993)

developed a practical ELISA for tracheal mite detection. Their technique

consists primarily of the following:

• Salt preservation. Alcohol preservatives destroy the mite components

detected by ELISA. Saturated salt preservative is made by dissolving

sodium chloride in boiling water until no more will dissolve (>30

percent weight by volume).

• A desalting step in an ELISA buffer solution is required before the

bees are homogenized.

• Whole, desalted bee samples (100 bees) are homogenized in ELISA

buffer for 60 seconds using a blender.

• A small, filtered sample is used in the ELISA analysis.

• The final steps involve a yellow color reaction and reading this color

intensity in an ELISA reader.

This technique is reliable for samples with a greater than 5-percent

infestation. For detecting lower infestations, one should analyze larger

number of bees in a sample or analyze the thorax or slices of thorax

instead of whole bees.

Tropilaelaps clareae

The distribution of Tropilaelaps clareae (fig. 16) is still restricted to Asia.

These mites parasitize brood. They have also been reported to infest

colonies with Varroa jacobsoni (Delfinado-Baker and Aggarwal 1987).

Female mites are about 1 mm long and 0.6 mm wide; the male is slightly

smaller. The mites are difficult to detect because of their small size and

their brownish color, but they can be seen under a magnifying glass or by

using a dissecting microscope to examine a brood comb suspected of

being infested. In the field, when the comb is jarred on a light-colored
34

Figure 16.

Tropilaelaps

clareae,

ventral view.

surface, dislodged mites may be seen moving on that surface. The mites

can be picked up with a fine brush moistened with alcohol.

Pests

Wax Moths

The greater wax moth, Galleria mellonella (fig. 17), is the most serious

pest of honeycombs. Comb damage can also be caused by the lesser wax

moth, Achroia grisella (fig. 17), and the Mediterranean flour moth,

Anagasta kuehniella. These moths are an especially serious problem in

tropical and subtropical climates, where warm temperatures favor their

rapid development. Female greater wax moths lay their eggs in a cluster,

usually in the cracks or between the wooden parts of the hive. The larvae

are the destructive stage. They actually obtain nutrients from honey, castoff pupal skins, pollen, and other impurities found in beeswax, but not

from the beeswax itself. Consequently, older combs are more likely to be

damaged than new combs or foundation.

Small Hive Beetle

Aethina tumida, the small hive beetle (SHB), was recently discovered in

the Southeastern and Mid-Atlantic states. In South Africa, where it was
35

first discovered in association with honey bee hives, the beetle is rarely an

economic pest. In the southern United States, it has caused thousands of

colonies to abscond, destroyed the marketability of honey, and rendered

combs useless.

Adult SHB are reddish brown and about 1/4 inch long, roughly one-third

the length of an adult worker honey bee (fig. 18). Adult beetles can be

found in brood cells, on the bottom board, or on the inner cover. The eggs

are laid in empty cells and are about half the size of honey bee eggs. The

larvae can get as large as 1/2 inch (fig. 19). They feed on brood, pollen,

and honey, then leave the hive, and pupate in the soil close by. Honey that

the larvae have fed upon becomes discolored and frothy and smells like

fermented fruit. If you have trouble identifying the insect, contact your

state apiary inspector or extension specialist for help. If help is not

available, send your sample in 70-percent alcohol to the Bee Research Lab

(see appendix C for address).

Figure 17. Top,

greater wax

moth, Galleria

mellonella.

Bottom, lesser

wax moth,

Achroia

grisella (not to

scale). These

species are

easily distinguished from

each other by

the shape of

the wings and

their comparative sizes.

The greater

wax moth is

about two

times larger

than the lesser

wax moth.
36

Figure 18. Small

hive beetle,

adults.

Figure 19. Small hive beetle, larvae.
37

Bee-louse

Braula coeca, or bee-louse (fig. 10), is actually not a louse but a wingless

fly that feeds on honey. No detrimental effect on adult bees has been

attributed to the bee-louse, but its larvae can damage the appearance of

comb honey. Adult B. coeca can be found on adult workers and queens. It

is important to note that B. coeca resembles Varroa jacobsoni in size and

color. However, Braula, being an insect, has six legs that extend to the

side. Varroa, an arachnid, has eight legs that extend forward.

Melittiphis alvearius

Melittiphis alvearius is a little-known mite that is associated with adult

honey bees but is not considered to be a pest. It is unlikely that M.

alvearius would be confused with other mites found in honey bee colonies. The adult female mite is ovate, flattened dorsoventrally, 0.79 mm

long 0.68 mm wide, brown, and well sclerotized with numerous stout

and spinelike setae. It is included here because it was found in California

during a survey for Varroa and because of increasing reports of its

distribution (Delfinado-Baker 1988).

Noninfectious Disorders

Noninfectious disorders can be the result of neglect, lethal genes, pollen or

nectar from poisonous plants, toxic chemicals (pesticides), and the like.

Most often, dead or discolored pupae result from a noninfectious condition. For a good review of noninfectious diseases, see Calderone and

Tucker (1997).

Neglected Brood

Normally, nurse bees feed and protect the brood. However, if there is a

sudden shortage of adult bees, the larvae and pupae suffer and may die of

chilling, overheating, or starvation.

Chilled brood

Chilling usually occurs in early spring when brood nests expand rapidly,

there is a shortage of adult bees to cover all the brood, and the weather

suddenly turns cold. Chilled brood are found most often on the fringes of

the brood area, and healthy brood remain at the center. Chilling can also

happen during cold weather following any sudden reduction of the worker

bee population. Chilled larvae and pupae are often yellowish, tinged with

black on segmental margins. They may also be brownish or black,
38

crumbly, pasty, or watery. In extreme cases, brood cells are punctured and

uncapped, and pupae are decapitated by the adult bees. It should be

remembered that decapitation can also be caused by the larvae of the

lesser wax moth.

Starved brood

Normally when there is a shortage of food in a colony, brood are removed

or consumed by the adult bees or both. However, when there is a sudden

shortage of adult bees to feed the larvae, the larvae starve. Affected larvae

are not restricted to the periphery of brood combs. The most striking

feature of starved brood is larvae crawling out of the brood cells in search

of food. Starved brood is almost always restricted to the larval stage.

However, emerging bees may starve if they were stressed as pupae by

chilling or overheating and if there are too few nurse bees to feed them

soon after they have chewed through their cappings. In these cases,

emerging adult bees usually die with only their heads out of the cells and

their tongues extended.

Overheating

The overheating of brood develops when there is a sudden loss of worker

bees to cool the colony during hot weather. Larvae that died from overheating become brownish or black and are watery; pupae have a black,

greasy appearance. Newly emerged adult bees may be wingless. Cappings

of brood cells can appear melted, darkened, sunken, and punctured.

Worker bees can overheat if they are confined in their hives during hot

weather without proper ventilation or access to water. Adult bees dying

from overheating crawl about rapidly while fanning their wings. They are

often wet, and their wings appear hazy. In some cases, an abnormally large

accumulation of dead bees may be seen at the hive entrance.

Genetic Lethality

Bees can also die from genetic faults during all stages of development,

usually without exhibiting symptoms of known diseases. However, drone

brood from laying workers and drone-laying queens often die with

symptoms resembling EFB but in the absence of known pathogenic

agents. Genetic lethality is the suspected cause of this condition.
39

Plant Poisoning

Poisonous plants can be a problem under certain conditions in limited

areas. If a plant’s nectar is poisonous, the symptoms of plant poisoning are

limited to the blooming period. If the poison is in the pollen, the symptoms may linger as long as the pollen remains in the combs. There is no

clear-cut method for differentiating between plant poisoning and pesticide

poisoning. The effects of plant poisoning are usually more gradual and last

longer than the effects of pesticide poisoning. Plant poisoning usually

occurs in the same geographical area at the same time each year, whereas

pesticide poisoning is indiscriminate. Some examples of plant poisoning

are listed below and in table 3. For a good review of poisonous plants, see

Skinner (1997).

Purple brood

Purple brood occurs when adult bees collect and use the pollen and nectar

from Cyrilla racemiflora (titi, southern leatherwood). This “disease” is

characterized by the blue or purple color of the affected larvae.

Paralysis

Aesculus californica (California buckeye) is probably the best known of

the poisonous plants in the United States. Field bees exhibit symptoms

similar to those of chronic bee paralysis; specifically, the bees are black

and shiny from loss of hair and they tremble. Also, either the eggs do not

hatch or the larvae die soon after hatching.

Milkweed pollinia

The pollen of milkweed (Asclepias spp.) is produced in pollinia (coherent

pollen grains) that are attached in pairs by a slender filament. When

removed from a flower, the pollinia resemble a wishbone with pollen

masses hanging from the ends. Honey bees become ensnared in the thin

pollinia attachment and free themselves by pulling the pollinia from the

flower. Honey bees often become seriously encumbered and cannot

remove themselves from the flower, or they may have difficulty flying and

crawl back to their hives with the pollinia still attached to their body parts.

Pesticide Poisoning

The most apparent indication of serious pesticide poisoning is the sudden

loss of adult bees. This loss is characterized by the appearance of many
40 Effect on brood

Table 3. Comparative symptoms in honey bees poisoned by toxic chemicals and selected plants

Toxic chemicals Adult Field bees die in or near Usually few larvae killed. Weakened or

hive or in field. Nurse Symptoms of starvation killed. Many

bees may also die. may be evident if adult dead bees near

Queens usually not population reduced hive.

affected. severely.

California buckeye Young Emerging young workers Eggs normal at outset; Weakened or

(Aesculus brood often deformed, pale. later fail to hatch or all killed. May be

californica) Some hairless and tremble. are drone eggs. Larvae many dead bees

Queens lay eggs at die soon after hatching near entrance.

reduced rate, cease, or and disappear. Little or Supersedure of

become drone layers. no capped brood; if queen may fail.

present, scattered.

Yellow jessamine Larva, Young workers affected Pupae die in cells and Slightly to

Gelsemium ( pupa, and and soon die. Old adults become mummified. to severely

sempervirens) young adult appear normal. weakened.

Loco plants Adult and Field bees die. Some be- Many cells contain Population

Astragalus ( spp.) pupa come black and tremble. dried pupae. dwindles.

Queen may die. Colony may die.

False hellebore Adult Many field bees die No effect. Field population

(Veratrum between plants and hive. lost.

californicum) Adults die in curled state.

Queens not affected.

Southern leather- Larva No effect. Many blue or purple Slight to severe

wood (Cyrilla larvae. Larvae die in cells weakening.

racemiflora) when nearly mature.

Source: Modified from Burnside and Vansell (1936).

Effect on adult

Effect on

colony

Stages most

affected Source of poison
41

dead and dying adult bees and sometimes pupae at the colony entrances.

In many instances, however, the bees are lost in the field before returning

to the colony. If the pesticide is brought back to the hive by the foragers,

the nurse bees die from feeding on contaminated honey or pollen and the

brood exhibits symptoms of neglect or poisoning. The symptoms of

poisoned honey bees often depend on the class of pesticide involved. For a

good review of poisoning from pesticides, see Atkins (1992).

Sample collection and preservation

Sampling must be done in a way that protects the sample from decomposition and further contamination. In the case of suspected pesticide poisoning, a sample of dying bees (at least 100) is placed in a clean, dry, preferably glass container. For comb containing pollen, honey, or brood, an

ounce or two can be placed in the same sort of container. Be sure to

include the date and time, location of hive, and name of beekeeper.

Samples can be stored for a few hours on ice but should be moved as soon

as possible to the freezer (the colder the better) until analysis. Leaving

samples unrefrigerated will render them useless. Check with your state bee

specialist or department of agriculture about laboratories capable of doing

pesticide analyses. The ARS Bee Research Laboratory does not do

pesticide analyses.
42

Appendix A. Microscopy

Most bee diseases can be diagnosed by observing the associated pathogens

or parasites with a compound light microscope. A stereoscopic (dissecting)

microscope is useful in identifying large parasites and pests.

Types of Microscopy

The following types of microscopy are referred to in this handbook:

• Bright-field. In bright-field microscopy, the microscopic field is

brightly lighted and the objects being studied appear dark. The

microscope should be equipped with two dry objectives (10and

40), an achromatic oil-immersion objective (100), and a good

light source. The bright-field microscope is used for most routine

microscopic procedures.

• Dark-field. The effect produced by the dark-field technique is a

black background against which objects are brilliantly illuminated.

This is accomplished by equipping the light microscope with a

dark-field condenser. Dark-field microscopy is particularly valuable

for examination of unstained microorganisms suspended in fluid.

• Phase-contrast. The phase-contrast technique employs controlled

illumination, which is obtained by special phase-contrast objectives

and a condenser assembly attached to a conventional light microscope. This system of controlled illumination reveals differences in

cells not discernible by other microscopic methods.

• Fluorescence. If a mixture of bacteria is treated with a solution of

fluorescent stain, the organisms that combine with or otherwise

take up this substance become fluorescent and can be detected in a

microscopic field illuminated with ultraviolet light.

• Electron. The electron microscope employs waves of electrons and

magnetic fields to produce the image, whereas the light microscope

uses waves of light and lenses.

Preparations for Microscopic Examination

Simple stain. Morphological studies of bacteria are generally done on

fixed and stained preparations. When a single dye is used, the process is

referred to as simple staining. Residue from a suspected cell is first mixed
43

with water. Then a drop of this suspension (smear) is spread on a glass

microscope slide over an area of about 2 c2.

The smear is dried and fixed under a heat lamp, or the smear can first be

air-dried and then heat-fixed by passing it, smear side up, several times

over a bunsen burner flame. The fixed smear is stained with a suitable

spore stain for 10 seconds. Carbol fuchsin, methylene blue, and safranin

are examples of stains that can be used. Enough stain should be placed on

the slide to cover the entire smear. The excess stain is then washed off

with water. 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 involves a fixed

smear that 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.

Modified hanging drop. The modified hanging drop technique (Michael

1957) can be very useful for differentiating brood diseases. 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 then heat-fixed by passing it,

smear side up, several times over a bunsen burner flame.

The fixed smear is stained with carbol fuchsin3

 or a suitable spore stain for

10 seconds. Enough stain should be placed on the cover glass to cover the

entire smear. The excess stain is then washed off with water. While the

smear is still wet, the cover glass is placed smear side down 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 formed in the

oil, and the organisms usually exhibit Brownian movement (see American

foulbrood, p. 4).

3Solution 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.
44

Negative staining. This is a technique by which, without staining, cells

are made readily visible in an otherwise dark film. The advantage of this

procedure is that the bacterial cells do not receive vigorous physical and

chemical treatment, which could result in distortion of some cells. Residue

from a suspected larva is thoroughly mixed with a drop of 5-percent (wt/

vol) aqueous nigrosin (India ink and Congo red are alternatives). This

suspension is then spread in a thin film across a glass microscope slide and

allowed to dry. Results: Bacteria appear transparent and outlined by the

dark background (see European foulbrood, p. 9).

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. Phase microscopy is

recommended for examining wet mounts, especially if an oil immersion

objective is required. Results: Organisms refract light and are therefore

visible on the slide.
45

Appendix B. Microinjection Techniques

To diagnose some diseases or determine toxic levels of sample materials,

it may be necessary to force-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 volumes as small as 1 microliter

(L).

Force-feeding

The microinjector can be used to introduce material orally into the midgut

(ventriculus) of larvae or to force-feed individual adult honey bees.

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 (fig. 20). When the actuating lever is depressed, a predetermined volume of material is pushed through the

esophagus and into the midgut, without physically damaging the larva.

After feeding, the larvae are placed in petri dishes lined with filter paper

and incubated at 34o C.

Esophagus

Microinjector

Midgut

Hindgut

Figure 20. Force-feeding a honey bee larva.
46

Adults. Individual adult honey bees can also be fed specific volumes with

the microinjector. The bees to be fed are held in a cage for about 4 hours

without food. The desired concentration of the substance to be fed 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 or

thorax, 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 the bees; the

gas makes the bees reluctant to feed and reduces their longevity. After

feeding, the bees are placed in small cages with a supply of 50-percent

sugar syrup and held in an incubator at 34o C.

Direct Injections

The microinjection technique can also be adapted for direct injections into

the body cavity of larvae, pupae, and adults. Care should be taken to insert

only the tip of the needle into the hemocoel and not to exceed 2 L per

bee.

Larvae. Injections are usually confined to 4- or 5-day-old larvae. Gently

grasp the larva between the first and second fingers and the thumb, and

hold it absolutely parallel to the needle. Using gentle pressure, puncture

the integument with the needle, expel the inoculum directly into the dorsal

blood vessel, and withdraw the needle slowly and steadily. To prevent

bleeding, avoid exerting any excessive pressure on the larva with the

fingers, particularly when withdrawing the needle.

Pupae. Pupae can be inoculated dorsally between the third and fourth

abdominal segments or through the propodeum of the thorax. After

injection, place the larvae or pupae in petri dishes lined with filter paper

and incubate them at 34o

 C. Any excessive bleeding will show on the filter

paper.

Pupae can also be left in brood combs and inoculated in the head capsule

(Wilson 1970). Expose the pupal head by removing the cell cap; then

insert the needle 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.
47

When bee longevity is a factor in the test, cold temperatures can be used

as an anesthetic. After injection, place the bees in small cages with a

supply of 50-percent sugar syrup and hold in an incubator at 34o C.

Collecting Larvae and Pupae

Honey bee larvae 3 to 5 days old can be readily obtained by removing a

brood frame from a colony and placing it horizontally above a towel-lined

tray in an incubator at 34o 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 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 removal from cells by mechanical

means.
48

Appendix C. Directions for Sending Diseased Brood

and Adult Honey Bees for Diagnosis to the ARS Bee

Research Lab

The diagnostic accuracy of any bee disease depends on the condition of

the sample. Mail the sample in a wooden or heavy cardboard box. The

sample can be loosely wrapped in a paper bag, paper towel, newspaper,

and so on, but avoid wrappings such as plastic bags, aluminum foil, waxed

paper, tin, or glass because they allow fungi to grow on the samples. The

ARS Bee Research Lab accepts samples from anyone and most samples

are processed within 1 working day.

Send all samples to

Bee Disease Diagnosis

Bee Research Laboratory

Beltsville Agricultural Research Center-East

Building 476

Beltsville, MD 20705

Include a short description of the problem, along with your name and

address. Plainly write your name and address on the outside of the

shipping container.

Samples of Brood

• The sample of comb should be about 4 square inches.

• The sample should contain as much of the dead or discolored brood

as possible.

• No honey should be present in the sample.

• If a comb cannot be sent, the probe used to examine brood should

contain enough material for tests. Wrap the probe in paper and send

it to the laboratory in an envelope. This method is unsatisfactory for

verification of sacbrood disease, which requires special antisera; we

rely on gross symptoms for this diagnosis.
49

Samples of Adult Honey Bees

• Send at least 100 bees in a sample.

• If possible, select bees that do not appear normal or that died

recently. Decayed bees are not satisfactory for examination.

• Bees submitted for the identification of mites should be placed in 70-

percent ethyl or methyl alcohol as soon as possible after collection.

These samples should be in leak-proof containers.
50

Appendix D. Identification of Africanized Honey Bees

ARS Bee Research Lab Protocol

The ARS Bee Research Lab uses FABIS, the Fast Africanized Bee Identification System Manual (Sylvester and Rinderer 1987) to identify European

bees. A full 26-character morphometric analysis is conducted on all samples

of honey bees not verified as European by FABIS. When the Bee Research

Laboratory declares a sample Africanized, identification is the result of the

full 26-character morphometric analysis and is definitive.

Submission of Honey Bees for Identification of Subspecies

The lab accepts samples from the Animal and Plant Health Inspection

Service, Plant Protection and Quarantine (APHIS-PPQ) and state regulatory

personnel. Preference is given to samples pretested according to FABIS and

identified as non-European. A sample of 100 bees should be submitted.

Honey bees submitted for identification should be placed in 70-percent ethyl

or methyl alcohol as soon as possible after collection. Place samples in leakproof containers and ship to

Honey Bee Identification

Bee Research Laboratory

Beltsville Agricultural Research Center-East

Building 476

Beltsville, MD 20705

Include a short history of the problem along with the sender’s name, address,

and telephone number. If results are urgently required, please advise the

laboratory by telephone (301–504–8205) of the shipment and send the

sample via express mail.

Addendum, May 3, 2005: The information on this page is no

longer correct. For current information on submitting Africanized

honey bees for identification, please visit this website:

http://gears.tucson.ars.ag.gov/morphometrics.html.
51

Appendix E. Reclassification of Bacillus spp.

Associated With Honey Bees

In 1992, the Subcommittee on the Taxonomy of the Genus Bacillus of the

International Committee on Systematic Bacteriology decided to undertake

a collaborative polyphasic4 study of the genus Bacillus. Among its

objectives were the establishment of minimal standards for the description

of species and the revision of nomenclature (ICSB 1993). 16S rRNA gene

sequence analyses have identified at least 10 phylogenetic groups in the

genus Bacillus. Five of these groups were reclassified as the new genera

Alicyclobacillus, Paenibacillus (Ash et al. 1993), Halobacillus,

Brevibacillus, and Aneurinibacillus.

To further complicate matters Heyndrickx et al. (1996) found P. larvae and

P. pulvifaciens to be similar at the infraspecific level. However, phenotypic and genotypic differentiation is possible and this is consistent with

their different pathologies. Therefore, they proposed reclassification of

P. larvae strains as P. larvae subsp. larvae and of P. pulvifaciens strains as

P. larvae subsp. pulvifaciens.

Bacillus spp. associated with honey bees have been reclassified as follows:

Bacillus alvei = Paenibacillus alvei

Bacillus apiarius = Paenibacillus apiarius

Bacillus laterosporus = Brevibacillus laterosporus

Bacillus larvae = Paenibacillus larvae subsp. larvae

Bacillus pulvifaciens = Paenibacillus larvae subsp. pulvifaciens

4 Polyphasic analysis = morphological and biochemical tests, a variety of

chemotaxonomic and genomic fingerprints, and DNA relatedness measurements.
52

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