7 Disease Inspection and Diagnosis

It is also not always a simple task to distinguish between the symptoms of AFB and other brood diseases and conditions in the hive. Identifying the sometimes subtle differences between symptoms of various bee diseases (called "differential diagnosis") requires both experience and regular refreshers. Fortunately, there are also laboratory tests which can be used to make AFB diagnosis less difficult.

7.1 Visual Inspections

To carry out a visual inspection easily and efficiently, it is important to work the hive in an orderly fashion. The inspection procedure should begin by removing all supers above the bottom box, and stacking them to one side on the up-turned lid. This divides up the bee population in the hive, and eliminates the potential for over-crowding of bees (if the hive is worked directly from the top box, a high proportion of house bees will be driven down during the inspection process, eventually crowding the bottom box and making it far more difficult to work).

Once the bottom super is exposed, one or two outside frames should be carefully removed from the box and propped on their side against the box. This creates a working space in the box that can be used when shaking bees off the brood frames.

7.1.1 Shaking Bees from Frames

Not all of the bees need to be removed to carry out a proper inspection. However, there should not be so many bees remaining that they make it difficult to see most of the face of the comb.

Taking the frame all of the way out of the box and then shaking the bees off is not recommended, since this usually results in more bees taking to the air (an action which can encourage robbing at certain times of the year). There is also a greater chance of either damaging or losing the queen because at least some of the bees will be shaken directly down onto the top bars.

7.1.2 Inspecting Frames

When the cell capping has been removed, or when a suspect uncapped cell is found, the colour and shape of the larvae/pupae should be observed.

Every brood frame in the hive should be inspected in the same way. Although a thorough inspection like this may take more time, both beekeeper experience and scientific trials have shown that inspecting all the brood frames in a hive pays important dividends, especially when it comes to identifying new, low-level infections before they have a chance to spread.

7.2 Field Diagnosis

7.2.1 Ropiness Test

To perform the test, insert a small stick (eg., wooden end of matchstick) into the suspect larva or pupa. Use the stick to mix up the brood remains, then remove the stick slowly. If the suspect larva/pupa has AFB, the tissues will draw out in a fine thread between 10 and 30 mm long (Plate 14).

The ropiness test is not always a definitive diagnosis for AFB, however. Diseased larvae or pupae in the early stage of decomposition and those that have started to dry may not "rope out" to any noticeable extent. As well, diseased larvae or pupae that have dried down completely to scale will not rope out, since there will not be any liquid material left in the cell.

It is important to use a dry stick with a rough surface to carry out the test. Remains of brood with AFB may not stick well to smooth or wet material like grass stems, and may therefore not rope out as far as they would normally.

7.2.2 Identifying Scale

The problem with identifying scale is that because the remains are flat and lie along the lower wall of the cell, they are not easy to see. The difficulty is compounded because cells are not actually perpendicular to the face of the comb. Cells actually slope upwards on about a 9^o angle.

If the frame is held directly flush, the scale is not usually visible because of the slope of the cells. If the face of the frame is turned too much on an angle, the scale is also not visible because the slope of the cell is very slight (Figure 10).

Figure 10. It can be difficult to see scale because cells slope upwards on about a 9^o angle.

To best see scale, the frame should be held by the lugs at either end of the top bar, and turned upside down (Plate 15). The sun, or a source of artificial light, should be positioned behind the observer, so that the largest amount of available light shines into the cells. The hands are then twisted at the wrists slightly downwards, rotating the face of the comb so that the largest surface of the lower wall of the cells is visible.

The lower walls of the cells are then searched for a distinctive curved shape (Plate 12). The shape resembles the rounded end of a bullet, and is identical to the head end of a sunken, moist, diseased larva (since AFB scale is the fully dried down remains of diseased larvae or pupae).

To better see the cell contents, the sharp corner of a hive tool can be used to carefully trim down the top side of the cell entrance. This will better expose the bottom wall of the cell where the scale is always found.

Sometimes comb in dead-out hives can be so degraded by mould, pollen mites or weathering, that it is almost impossible to see the outlines of larval scale in the cells. In such cases, a thorough search of all the combs should be made in an effort to find the thin hair-like structure that is the remains of the tongue in pupal scale (Plate 13). The pupal tongue can be regarded as a definitive field diagnosis for AFB, since no other disease will produce this symptom.

American foulbrood scale cannot be removed from cells without breaking apart the cell wall. The pupal tongue, however, is very fragile, and can easily be broken off with the sharp corner of a hive tool.

7.3 Differential Diagnosis

Effective differential diagnosis often relies on comparing a set of symptoms, rather than concentrating on single symptoms, and looking at a range of larvae and pupae in the hive. If there is any question about the symptoms of a particular larva or pupa, the entire colony should always be checked for further symptoms. If a definitive symptom is found, the beekeeper should still confirm the diagnosis by searching for 3 or 4 more diseased larvae or pupae elsewhere in the hive.

The following table compares the distinguishing features of AFB and the other brood diseases and abnormalities:

Table 2: Distinguishing Features of Brood Diseases and Abnormalities

7.3.1 Sacbrood

Cells containing sacbrood can be either capped or uncapped. If the cells are capped, perforations in the cappings appear in the same way as for AFB, since in both cases the perforations are created by house bees that have detected diseased larvae underneath.

Decaying larvae killed by sacbrood tend to remain more rounded in shape than those killed by AFB, at least in their early stages of decomposition. The larval skin of sacbrood infected larvae become tough and plastic-like (Plate 16), and retain the segmentation visible in healthy brood. AFB infected larvae do not show this clear segmentation.

At the early stage of decomposition, larvae infected with sacbrood can sometimes be removed with a stick or twig as whole larvae (Plate 17), and will hang in a sack-like fashion (hence the name "sac" brood). If the larvae is punctured, a watery fluid will come out. AFB infected larvae do not display such symptoms.

Sacbrood infected larvae go through a series of colour changes, from yellow, to brown, to grey, and finally to black. AFB infected larvae also go through a series of colour changes, but the change is from light coffee-brown colour to darker coffee-brown colour, and finally to black. Sacbrood infected larvae at the brown colour stage are the ones most often confused with AFB infected larvae.

In sacbrood infected larvae, the head-end of the larvae usually remains darker than the rest of the body, and the head-end is often raised (Plate 18). AFB infected larvae do not generally display two-tone colouration, although at the onset of the final drying down of AFB larvae into scale, the head-end of the larvae can become drier than the rest of the larvae, giving it a darker appearance. The head-end is not raised in AFB infected larvae, however.

Unless house bees remove them, sacbrood infected larvae eventually dry down to black-coloured scale, which are similar in appearance to AFB (although no pupal scale or associated tongues are ever found). Unlike AFB scale, sacbrood scale can easily be removed from the cell wall, often in one piece. Sacbrood scale are also generally found only in colonies suffering bad infections. Such infections are rare.

Beekeepers often use the ropiness test when they find sacbrood infected larvae in the brown colour stage (after the larvae have collapsed, but prior to drying down to scale). It can be difficult to make a visual diagnosis of such larvae, since the symptoms so closely resemble AFB. Generally, sacbrood infected larvae at this stage will not show the degree of roping associated with AFB. However, under some circumstances sacbrood infected larvae will show some roping. The roping is probably caused by secondary invader bacteria feeding on the larval remains.

Because ropiness can also be variable in AFB infected larvae, it is important to look for other, contrasting symptoms when making a differential diagnosis for sacbrood. The remainder of the brood should be checked carefully for more definitive AFB symptoms (and especially pupal scale). If no further symptoms can be found, a sample of the suspect (roping) larva should be taken for laboratory test (see Section 7.5).

7.3.2 Chalkbrood

The only symptom resembling AFB is the presence of perforated cappings, which are identical in appearance to both sacbrood and AFB, since once again with chalkbrood the bees chew the cappings off when they identify a diseased larva beneath.

Chalkbrood infected larvae turn a vivid white which is much whiter and "chalky" in appearance than the colour of healthy brood. At this stage the larvae have a furry surface because of the fungal strands growing on the dead larvae. If the larvae are removed, they are moist, hexagonal-shaped and swollen. In most cases, the head-end of the larva appears clearly as a dark spot in the remains (Plate 19). When chalkbrood infected larvae are removed with a stick, they never rope out of their cells.

Chalkbrood infected larvae later dry out and become hard chalky lumps called "mummies". These remains are mostly white, although some mummies turn partially or completely grey or black (Plate 20). The colour change is the result of fruiting bodies. These bodies produce the fungal spores that are the infective stage of chalkbrood disease.

Chalkbrood mummies are often removed by house bees and are sometimes found either on the bottom board or in front of the entrance of the hive. Combs with heavy infections of chalkbrood will rattle when the mummies are sufficiently dry. At this stage the mummies can easily be removed just by shaking the comb.

AFB infected larvae do not turn either chalky white or grey in appearance, and do not dry down into easily removed mummies. The only condition in the hive that closely resembles chalkbrood is mouldy pollen. However, mouldy pollen is cylindrical in shape and is easily pulverised between the fingers, whereas mummies are more elongated, are much flatter, and are not easily crushed.

7.3.3 European Foulbrood

The visual symptoms of EFB can be mis-diagnosed as AFB. For instance, in advanced stages of EFB, larvae may die after the cell has been capped. In such cases, the brood pattern may appear patchy and some cappings may be sunken or perforated. These symptoms are also characteristic of AFB. Larvae affected by both diseases also eventually dry down to form scale.

There are a number of distinctive differences in the symptoms of these two brood diseases, however. A major difference relates to the stage of development at which the larva succumbs. In the case of EFB, death always occurs prior to the larva laying out fully along the bottom wall of the cell (pre-pupal stage). Death usually occurs when the larva is still curled in a c-shape in the cell (Plate 21). There are circumstances, however, when the larva dies in the transition between this c-shape and full extension. In such cases, the larva appears to be twisted, in a cork-screw shape, from the bottom through to the top of the cell (Plate 22).

AFB infected larvae and pupae are never found in either the c-shape in the bottom of the cell, or twisted in a cork-screw shape. AFB infected brood are always found in the prepupal or pupal stage. EFB infected brood are never found in the pupal stage, and the remains of tongues are never present.

Another distinctive difference is the colour of brood affected by the two diseases. In EFB, infected larvae turn from the normal pearly-white colour to yellow. Outlines of the larva's tracheae also appear as light lines amongst the yellow tissue (Plate 23). Tracheal outlines are never prominent in the larvae affected by AFB.

EFB infected larvae occasionally rope out when a stick is inserted into the cell and slowly withdrawn. The roping is caused by the presence of other bacteria in addition to EFB that are feeding on the dead larvae.

When the remains of EFB infected larvae dry out, a distinctive c-shape scale is formed. Unlike AFB scale, this scale can easily be removed from the cells.

The smell of EFB infected larvae is sour and urine-like, and is not similar to the fishy smell associated with AFB. However, because of the variability in people's ability to detect and differentiate odours, smell should never be used to diagnosis either disease.

Although EFB is currently not found in New Zealand, beekeepers should still be aware of the symptoms of the disease, and should always send samples of suspect brood to a laboratory for diagnosis. Contact the Ministry of Agriculture for details. Beekeeper vigilance is the first line of defence in keeping New Zealand free of unwanted honey bee pests and diseases.

7.3.4 Half-moon Syndrome

The features of half-moon syndrome closely resemble those of EFB. These include patchy brood patterns, perforated cappings (in advanced stages), curled larvae (either c-shaped in the bottom of the cell, or twisted in a "half-moon" shape from the bottom to the top of the cell), yellowish colouration and tracheal outline, and a sour, urine-like smell. Half-moon syndrome can also be mis-identified as AFB.

The only major difference in appearance between half-moon syndrome and EFB is the presence of multiple eggs in cells (Plate 24). In half-moon syndrome, this multiple egg laying can be found in many worker cells, with the eggs attached in chains, rather than all fastened to the bottom of the cell (as is the case when the colony has laying workers). Multiple egg laying is not associated with EFB or AFB.

In hives with advanced cases of half-moon syndrome, a high percentage of drone brood (including capped brood) can be found in worker cells, with very little worker brood present. Beekeepers generally believe that such a symptom is the result of a poorly mated or old queen, and re-queen the hive. Re-queening usually alleviates half-moon syndrome.

Because the distinguishing features of half-moon syndrome so closely resemble the symptoms of EFB, beekeepers should always send a sample of affected brood to a laboratory for diagnosis. Contact the Ministry of Agriculture for details.

7.4 Inspection Training and Refreshers

Experience has shown that beekeepers with little or no AFB in their outfits can lose their ability to make differential diagnoses of brood diseases because they do not have the opportunity to periodically see AFB in the field. This is especially the case in larger beekeeping outfits, where staff may not have been working for the business at a time previously when AFB was a more serious problem.

One of the best opportunities to obtain training and refreshers in AFB inspection is to attend the annual AFB Elimination Field Day held by local branches of the NBA. These workshops allow newer beekeepers to learn from those who have been working in beekeeping for a longer period of time. They also provide an opportunity for all beekeepers to trade experiences and techniques used in AFB elimination.

Another good opportunity to obtain training is to attend an NBA-sanctioned course in AFB recognition and destruction, and to take the competency test required for Approved Beekeeper status under the AFB PMS. Beekeeping businesses should use these courses as a means of providing staff with a basic understanding of AFB identification in a structured learning environment.

It is also a good idea to retain one or two frames showing AFB symptoms for future reference (to retain these frames, a permit must be obtained from the Management Agency). Frames for this purpose should be wrapped in newspaper (for absorbency), and put in a large plastic garbage bag secured with a twist-tie or tape to exclude moisture. The wrapped frame should be kept in a freezer (to control fungal growth) and removed several hours prior to use.

A good time to look at these frames is at the beginning of the beekeeping year, just before the first inspection round. Scrapping cappings off, looking at cell contents, checking for scale, and performing the ropiness test, provides an opportunity to "get one's eye in" after the winter period.

7.5 Laboratory Diagnosis of AFB

Two tests are used. The first is a microscopic analysis, and is only used for samples of suspect larvae or pupae.²⁸ The test involves making a smear on a microscope slide, fixing the smear to the slide using heat and carbol fuchsin stain, and then examining the smear for AFB spores using the oil immersion objective of a microscope. The total magnification required is 1000x.

The second test is a culture test, and involves growing out bacterial cultures of AFB. The test can be used with samples of adult bees, honey, or suspect larvae/pupae, and can be carried out in a home laboratory.²⁹ A dilution of the sample is prepared, and then heated at 92^oC for 20 minutes to kill all competing bacteria, but not the AFB spores. A small amount of the liquid is applied to a petri dish containing sterile brain heart infusion agar, and then the plate is incubated at 37^oC for 3 days. The plate is then inspected for the presence of AFB bacterial colonies.

7.5.1 Deciding What to Test

Sample types include:

1. Confirmation of Diagnosis - required because there are one or more AFB-like symptoms in the hive, and a confirmed diagnosis is needed. The material tested should always be larvae or pupae actually showing the AFB symptoms. Adult bees or honey should not be tested for confirmation of diagnosis.
   
2. Surveying the Disease Status of Colonies - required because colonies may have been exposed to AFB, but are not currently exhibiting any disease symptoms. The best material to have tested is adult bees taken off brood frames. Honey or larvae/pupae should not be tested.
   
3. Surveying the Disease Status of an Apiary or Group of Apiaries - required to identify any potential disease problems in the future when no disease is currently being found. Composite bee samples can be used, consisting of 30 bees from each hive in an apiary, with all the bees put into the same container. Honey can also be tested from the extracting tank, although it may be harder to relate the results back to the correct apiary.

7.5.2 Collecting Samples

7.5.2.1 Adult Bees

The following procedure should be followed when taking adult bee samples:

• Open the hive and either find the queen or thoroughly inspect a brood comb to ensure the queen isn't present.
  
• Prop the brood frame up against the hive.
  
• Open an unbreakable plastic jar and scoop it along the side of the frame to trap the bees inside.
  
• Collect at least 30 bees. Put the lid on before the bees can fly out.
  
• If a small plastic bag is used instead of a jar, hold the bag open with both hands and scrape up the comb to collect the bees. Twist the bag closed before the bees can fly out.
  
• Don't worry about killing the bees. They will quickly suffocate in the sample container.
  
• Samples must be kept cool and dispatched to the lab on the day they are collected, or frozen as soon as possible after collecting (preferably within 12 hours). Otherwise, non-AFB bacteria may grow in the sample and interfere with the laboratory test.
  
• Do not send samples to the lab on a Friday or on the day before a public holiday, since the samples may grow non-AFB bacteria during the delay before delivery to the lab. *

7.5.2.2 Larvae/Pupae

The following procedure should be used to take samples of larvae or pupae exhibiting AFB symptoms:

• Remove the suspect larva or pupa from the cell with a matchstick.
  
• Place the larva/pupa in an unbreakable plastic jar or plastic bag.
  
• Put samples from one hive in one jar/plastic bag. If possible, collect at least three larvae/pupae that are showing similar symptoms.
  
• Take further samples from the hive until the jar/bag contains a representative sample of the AFB symptoms present in the hive.
  
• Do not use the same jar/bag for samples from more than one hive, since this will make it difficult to identify the sample back to the individual hive.
  
• If the matchstick is to be included in the sample jar/bag, break off the sulphur end of the match first (since the sulphur may contaminate the sample). If the matchstick is not to be included, dispose of the match stick by putting it back into the hive. It's a good idea to put the match stick back in the suspect cell, since this will make it easy to find the cell again once the lab results are received.

7.5.2.3 Comb

Comb samples are not recommended because the laboratory may not be able to identify the individual suspect cells on the piece of comb. Comb is also subject to crushing in the post.

Comb should only be sent where there is no easily removed larval material (eg., dried remains from a dead-out colony). In this case, it is best to take an entire frame as a sample.

7.5.2.4 Frames

The following procedure should be used to prepare samples of brood frames showing AFB symptoms:

• Wrap the frame in a sheet of newspaper. This will help to absorb any moisture which might otherwise leak out during mail shipment.
  
• Put the wrapped frame in a plastic rubbish bag. Tie off the end, then wrap the frame again in heavy wrapping paper.
  
• Make sure the frame is mailed as soon as possible, since the comb will begin to grow mould that could interfere with the diagnosis.

7.5.2.5 Honey

Honey can be collected from individual hives, the extraction tank, or from a drum, and in either liquid or granulated form. Put at least 400g in an unbreakable plastic jar. If honey is taken directly from a hive, the comb scrapings do not need to be removed from the sample.

7.5.3 Marking the Hive

7.5.4 Labelling the Sample

• The Beekeeper Registration Number (eg., D0001), followed by a forward slash (/), and then a code number chosen for the hive (eg., A1). The result will look something like: (D0001/A1). If a plastic bag is used, the outside of the bag should be marked with this code using a marking pen.
  
• For a frame sample, the information (eg., D0001/A1) should be written on a small piece of paper and put into the plastic bag before tying the bag off.

7.5.5 Preparing an Information Sheet to Accompany the Sample

• Name, address and telephone number, printed to ensure legibility
  
• Number of sample jars/bags being sent, and the number of hives being sampled
  
• The code number used to identify each hive and mark each sample
  
• Description of the AFB symptoms (if appropriate)
  
• Type of test required

7.5.6 Interpreting Laboratory Results

To be useful, the results need to be interpreted. The meaning of the result will depend on the type of material submitted.

7.5.6.1 Larvae/Pupae Samples

If a larvae/pupae sample returns a positive test for AFB, this means the colony the larvae/pupae was taken from had AFB when the sample was taken. The colony should be destroyed even if no other diseased larvae or pupae can be found.

7.5.6.2 Adult Bee Samples (Individual Hive)

Negative Results

AFB spores do not germinate well on plates. The number of colonies therefore provides only a relative measure of the number of spores present in the sample.

A negative result does not necessarily mean there are no AFB spores in the sample, only that there are too few to be detected. However, if there are too few spores to be detected, it is unlikely that the spores will cause an AFB infection in the sampled colony.

As well, bacterial culture tests are not 100% accurate. An adult bee test may occasionally test negative for AFB spores even though the colony contains a few diseased larvae or pupae.

The test results only relate to the time that hive was sampled. Just because the colony didn't have AFB when the sample was taken, this doesn't mean that it will not have AFB at some later date.

Positive Results

A positive result may or may not mean that the sampled colony has AFB. For instance, one of the bees in the sample could have drifted from an AFB hive that was nearby the hive that was sampled.

In 1991, live bee samples were tested from 1800 colonies belonging to 7 different beekeepers. When the hives were inspected immediately after the test, only 35% of the colonies testing positive for AFB spores were found to contain obviously diseased larvae or pupae.³⁰ However, more colonies in the positive category showed AFB symptoms during later inspections.

Counting the number of AFB bacterial colonies per plate can provide a better indication of hives likely to contain diseased larvae/pupae. Table 3 shows that the greater the number of colonies per plate, the greater the probability that the colony already shows visual signs of AFB.³¹ (Note: this study used 60 ul of sample material applied per plate).

Colonies/plate	Percent of colonies showing visual AFB symptoms
0	1%
1	1%
2-10	7%
11-100	16%
>100	82%

Table 3. Relationship between the number of bacterial colonies per plate and the percentage of colonies likely to exhibit visual disease symptoms over the following year.

If an adult bee test is positive, every brood frame in the hive should be checked thoroughly as soon as possible. If diseased larvae or pupae are found, the colony must be burned. If no diseased larvae or pupae are found, the colony should be marked and quarantined for one full year. At the end of that period, another complete brood check should be carried out together with a culture test using adult bees. If a hive tests positive again, but does not show visual signs of the disease, it should be left in quarantine for another full year.

During the quarantine period, no hive parts should be removed from the colony or placed on any other colony. It is also worth moving positive test colonies to a separate site where they can be inspected more often.

7.5.6.3 Adult Bee Samples (composite samples from a number of hives)

It is more difficult to interpret the results of composite samples where bees from a number of hives have been tested at the same time. Because of the dilution factor of the bees from the clean colonies in the composite samples, composite tests are more likely to fail to detect the presence of an infected colony.

If there is only one AFB colony represented in a composite sample, then the results of the test can be interpreted by dividing the colonies/plate in Table 3 (above) by the number of colonies making up the composite sample. This will give the probability of the group of hives having one hive with AFB.

There may be several colonies with low spore counts represented in the composite sample, however, that when added together give a high spore count in the composite sample. For this reason, all that can be said with certainty is that a negative result in a composite sample indicates that the group of hives is unlikely to have AFB, and a positive result indicates that there may be one or more colonies in the group that have AFB. All hives in a composite sample should therefore be given a complete visual examination. If this fails to identify the AFB hive, then the hives should be retested individually.

7.5.6.4 Honey samples

Honey samples are less suitable for detecting AFB hives than adult bee samples. This is because the honey may have been collected before the colony developed AFB. The relationship between spore levels in honey from an individual hive and the probability of the colony exhibiting AFB disease symptoms is unknown. Any hive that does test positive should be treated in the same way as a colony testing positive in an adult bee test (see above).

Tests of extracted honey tell even less about the hives the honey came from. Whether a positive sample with a high spore count came from a single hive with a very bad case of AFB or a number of colonies with light infections cannot be determined. If a positive sample can be traced back to a particular apiary, however, it can be useful in identifying which colonies should be give be given a visual examination.

If a large number of extracted honey samples test positive, it suggests that there might be a large problem in the beekeeping outfit.