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Diagnosis of Honey Bee Diseases

Bee Disease is unfortunately a fact of life, but many beekeepers pay scant attention to learning about disease recognition. It is obviously not a popular topic as whenever the subject of a lecture is 'disease recognition' or diagnosis, the lecture is poorly attended.

An obious line of empty cells caused by foundation wire

Whenever a beekeeper is manipulating his (or her) bees they should be vigilant to any signs of disease, but this vigilence on it's own is not enough, two inspections should be carried out on each colony every year, one early in the season and one late, the sole objectives of these two manipulations is examination for signs of disease and the reason that the inspections need to be specifically allocated to this task is that the method of disease inspection is not quite the same as would normally be carried out if we were just looking for swarming preparations or whether the colony has enough room.

Disease inspections require that every frame with brood in a colony is looked at and that such observations should be made with as few bees as possible adhering to the frames. In normal manipulations it is common to close down a hive once our reasons for opening it are satisfied by suitable observations, but as disease may be present in just one cell or a single larva, then every occupied or sealed cell must be looked at.

Beekeepers must be able to recognise bee diseases and parasites and to differentiate the more serious diseases from those that are less important. Brood combs of healthy colonies usually exhibit a solid and compact brood pattern. Almost every cell of the comb central area will contain an egg, larva, or pupa. The cappings of sealed cells will be uniform looking and of an oatmeal colour, each capping will be convex, but not domed or bullet shaped. If you have a few cells that are vacant, but they follow the line of frame wiring, it may well be the intrusion of the wire into the base of the cell that causes the cell not to be used, but as shown in the image at right, the regularity of ocurance of such cells will be obvious to you.

In contrast brood combs of diseased colonies usually have a spotty brood pattern (pepperbox appearance that is called shotgun brood in USA), the cappings are often darker and concave, some may be perforated. The capped cells may be more varied in colouration as well as being generally darker. The combs may contain scales caused by the dried remains of larvae sticking lengthwise on the lowest side of brood cells, these can be difficult to see, so angle the combs in order that sunlight can illuminate the lower cell surfaces.

However... If you open a hive and you see nice even brood patterns and light oatmeal coloured cappings, do not be complacent. You still need to be vigilant and look at every cell as it is easy to be lulled into a false sense of security by the apparently good combs. I would say it is easy to miss a single diseased larva or cell under these circumstances.

Regional Bees Inspectors and their seasonal counterparts will use a technique for freeing the frames of bees that I will describe, but I have a personal dislike of this method, I have seen it performed badly, with many bees damaged and indeed frames broken by heavy handed application of the process, which I found unnecessary with my own stock as they were much less excitable than the majority of bees found in UK.

Method of examination... This text is a direct copy of DEFRA instiuctions.

Remove the outside comb, which is unlikely to contain brood, and lean it against a front corner of the hive. You will then have room to work.

Take each comb in turn, and holding it by the lugs within the brood chamber, give it a sharp shake. This will deposit the bees on the bottom of the hive without harming them, the queen or brood.

Scan the brood area of each side of each comb that contains brood in any stage, look for areas that exhibit a different colour either in cappings or in the larvae themselves. Look for evenness of capping and pay particular attention if there is a perforation in a capping. However, it should be noted that the perforations we are looking for are small and irregular, not the roughly circular, large area associated with bald brood. A further distinguishing mark for bald brood is that the edge of the hole in the capping may be raised like a very short chimney.

American 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?.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

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.

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, mA. m. melliferaals, 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.

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? m long  2? 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). 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?rrivee and Hrytsak 1964).

Other Protozoa

Gregarines
Four gregarines (protozoans of the order Gregarinida) are associated with honey bees: Monoica apis, Apigregarina stA. m. melliferaeri, Acuta rousseaui, and Figure 8. Cross sections of Malpighian tubules. Top, healthy tubule. Bottom, tubule containing cysts of Malpighamoeba mellificae. 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.

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 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).

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 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:

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

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.

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.