A SURVEY ON AMERICAN FOUL BROOD IN CHAHARMAHAL AND BAKHTIARY PROVIANCE

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A SURVEY ON AMERICAN FOUL BROOD IN CHAHARMAHAL AND BAKHTIARY PROVIANCE

FROM JAN 2003 TO MAR 2004

Introduction:

Foul brood :

The term ‘foul brood’ covers two diseases of the honey bee larvae, one known as American foul brood (AFB), and the other European foul brood(EFB). The names bear no relation to the geographical distribution of the diseases .

Cause

American foul brood is caused by a spore-forming bacterium called Paenibacillus larvae.

 Paenibacillus larvae.

Young honey bee larvae become infected when the consume P. larvae spores in their food. The spores germinate in the gut; bacteria then move into the tissues, where they multiply enormously in number. Infected larvae die after their cell is sealed, and millions of the infective spores are formed in their remains, These remains dry to form ‘scales’ which adhere closely to the cell wall and cannot easily be removed by bees. Consequently brood combs from infected colonies are inevitably severely contaminated with bacterial spores.

spore of  Paenibacillus larvae

     American foul brood (AFB) is one of the most destructive brood disease in our beekeeping industry . In our province (chaharmahal and bakhtiary ) it is one the cause of deaths at apiaries . For this reason , we tried to investigate the spread rate of this disease in our province ,from JAN 2003 to MAR 2004 for this reason we collected 1860 sample, honey and larvae from 10 percent of the colonies(during14 months) .The result showed that 93 cases of honey and 33 cases of larvae were infected with bacillus larvae. The ifection was determinated by using hanging drop method (MHD) and microbial culture in Colombid blood agar and MYPGP agar .The honey was cultured in the same agar .The  cause of the disease is a gram positive, spor  forming bacillus called Paenibacillus larvae . This bacillus was identified by white in 1920.  The spores are very resistant to extremes of heat and cold, and to disinfectants. They retain their powers of germination for many  years in honey, in old combs kept in store, or in derelict hives, skeps or boxes.   Once a colony is infected the disease will progress until           most of the brood is affected. The colony then becomes unable to replace the ageing adult bee population, causing it to become weakened, and finally to die out. The disease may develop for months before the colony succumbs, and death may occur at any time of the year.

SIGNS OF AFB:

     AFB affects only sealed brood. When infected larvae die with in the sealed cell, the appearanceof the cell cappings changes.· Cappings become sunken and perforated when adult bees nibble holes in them. These perforations tend to be jagged and irregular in shape.

    Some cappings may become moist or greasy looking and slightly darker in colour than other cells at first only very few cells may show signs of disease, and the colony will appear normal in other respects.Eventually much of the sealed brood will become affected by the disease, causing a patchy or ‘pepper pot’ brood pattern. There may then be an unpleasant smell.At the sunken capping stage the dead larval remains are light to dark brown in colour, and have a slimy consistency.

If a matchstick is inserted and slow withdrawn,the remains can be drawn out in brown,mucus-like thread or ‘rope’ 10-30mm longThis is called the ‘ropiness’ test and is a reliabletest for AFB.

Further drying leads to the final stage, which is a very dark brown rather rough scale lying on the lower side of the cell and extending from just behind the mouth of the cell right back to the base.

The scales can be detected if the comb is held facing the light: they reflect the light from th rough surfaces and can easily be seen, even when their colour is almost the same as the comb itself.Further drying leads to the final stage, which is a very dark brown, rather rough scale lying on the lower side of the cell and extending from just behind the mouth of the cell right back to the base.

The ropy condition is followed by a tacky stage as the larval remains in the cell gradually dry up and the colour changes to dark brown.

The proboscis of dead pupae may sometimes remain intact, protruding upwards from the bottom edge of the cell.

Spread :

The beekeeper is the chief spreading agent of the disease. If combs, honey or hive equipment are transferred from an AFB-infected colony to a healthy colony, it becomes infected. The disease is also transmitted by bees robbing honey from infected colonies. Swarms from infected colonies may also carry infection with them and become diseased after they are hived.

Methods:

    In our province, from JAN 2003 to MAR 2004 honey and larvae were collected from 99 apiaries .

The aim of this research was to examine the larvae died due to AFBAnd the scales of the dead larvae .

The Laboratory Examination:

   The determination of pollution of broods has done through Hanging Drop(M.H.D) and bacterial culture of honey and brood in MYPGP Agar and colombia blood agar.

Modified Hanging Drop (MHD) :

    For this purpose Modified Hanging Drop (MHD) , one of the safest methods in this diagnosis of honey bee and larvae diseaseswas used.

In this method, dead larvae remains are spread over the lamella .After that they are fixed by heating and dyed with fushin zil .Then they are washed and , when wet, tyeyare spread on ascale on which is immersion oil .They are placed in such a way that the smear is in contact with the immersion oil.

   Finally, they are examined with objective lens 100x.In his case B. Larvae spores have Browny movment in water drops.This test is negative for B.pulvifacious,B,Alvei and B,Apiarius,

   AFB is a notifiable disease and is subject to official control by a programme of apiary inspections and compulsory destruction of infected colonies. For confirmation of AFB a sample (eg brood comb, suspect larvae in plastic tube) is sent to the  laboratory where larval remains are examined microscopically for the presence of the causative bacteria.

Bacterial culture:

In this study we used MYPGP AGAR and Colombia blood agar .

Culture—and catalase test are as follow::

MYPGP-AGAR :

M:Mueller- Hinton brooth             1.0%

Y: Extract of yeast powder             1.5%

P: Po4K2h                                      0.3%

G: Glucose Monohvdrate               0.2%

P: Pyruvic acid –Salt                      0.1%

Agar                                                  2%

All the materials are poured in a beaker and distilled water , added until the volume is 100cc then they are heated for about 50 minutes until the agar is even .After that they are put in an autoclave at 121c and 15p.s.i fot 20 minutes until they becam sterilized. The environment is ready to be spread after it is cooled .

COLOMBIA BLOOD AGAR:

39gr of colombiad blood agar is put in distilled water ,and heated completely.then it is sterilized in an outoclave 121c for 15 minutes .When the temperature is 50c 7% stril sheep blood is added to it .Then they are divided into sterile petridishes .

Honey Culture:

Honey culture is one of the  most effective ways of screening in the examination   of the beehaives. By the use of the information obtained in this way, we can identify the disease.Factor, ie.Bacillus larvae one year before the symptoms emerge.The factor can be separated from the beehive and economic control programs can be designed.In this way ,we can decrease the mortality rate and hece honey culture is of prime importance.

Methos: Honey culture

First 5 gm of honey is put in two sterile measuring beakers, 5cc of distilled water is added to each and the sulotion is thoroughly shaken.Next, the samples are placed in a ban marie(bach water) .After incubation at 90cdrgree  for 5 minutes,0/4cc of each sample is poured in four petridishes having Mypagar and four petridishes having colombiad blood  agar . After that the petridishes are put in an incubator at 37c degree .Next day , the petridishes are turned 180 degree .The colonies of bacillus are smaller thane 2 mm and they donot have a light color . Their catalase is negative . Spiral threads are observed in necrosin test.

Larvae and scaly larvae remain culture .Asterilised capped test tube having crystal shots was chosen.

Larvae and scaly larvae remain culture .   Asterilised capped test tube having crystal shots was chosen about 2cc distilled water was poured into it .A number of suspect larvae and their dried scales ,which were collected from the depthse of stones, were put in the tube and were thoroughly shaken .The solution obtained in this way was used in the above mentioned culture agar  After that the prtridishes are put in an incubator at 37c degree .Next day , the petridishes are turned 180 degree .The colonies of bacillus are smaller thane 2 mm and they donot have a light color . Their catalase is negative . Spiral threads are observed in necrosin te

Then we did the tests as the temple below:

Catalase test :

The spors of bacillus larvae develop fally in Mypgp-agar and co;ombia agar after four days.When a drop of hydrogen peroxide(3%) is poured on them , there is no change . When ther are a lot of bubbles ,the bacillus is not B.larvae since B.larvae has negative catalase.

Reduce the interchange of material between colonies or from other apiaries :
a). Do not feed honey from an unknown source.
b). Let your bees draw their own comb. Keep control over where comb is and whether bees have access to it. Don’t, for example, leave brace comb lying around the apiary because if it is a source of infection you’ll never find it.
c). Prevent robbing and close colonies that have died until the cause of death is known.

d). Position hives to reduce drifting between colonies e). Be aware that hive tools, gloves, clothing and even the smoker can transfer disease-carrying material. Introduce swarms with care, never hive them on to drawn comb and check the disease state by examining the brood as soon as possible. Know your locality and the risk to which you are exposed.

Be cautious with second-hand equipment, sterilising it before use. Find out its provenance and inspect it carefully.As with other bacterial pathogens antibiotics can be used. These act by preventing the reproduction and growth of the bacterium, for example by inhibiting the formation of the cell wall. None are capable of acting through the thickened wall of the bacillus ‘spore’ and for this reason are said to ‘mask’ the infection, the disease reappearing when treatment stops.

Oxytetracycline is in current use in many countries (known by the trade name Teramycin or TM25). Resistance to tetracyclines is common in human pathology but so far rare as far as bees are concerned. This may be due to the speed with which the compound breaks down when exposed to warmth, moisture or sunlight, until recently OTC was administered as a dust with powdered sugar.

There can certainly a residual contamination of honey when antibiotics are used on bees and a period of eight weeks must pass before honey can be used.

Control :

AFB is a notifiable disease under the Bee Diseases Control Order 1982 and is subject to official control by a programme of apiary

inspections and compulsory destruction of infected colonies. For confirmation of AFB a sample brood comb is sent to the laboratory where larval remains are examined microscopically for the presence of the causative bacteria.

Infected colonies are destroyed by burning under the supervision of a bee inspector. The bees are killed, and together with the combs are burned in a deep pit. Hives and appliances can be sterilised by thoroughly scorching them with a blowlamp. Gloves, overalls, footwear and the smoker are washed in hot soapy water.

Methods of control of AFB using antibiotics that are used in some countries are not effective, as they suppress signs of the disease without eradicating it

Results:

American foul brood is one of the most destructive disease throughout the world.In different countries ,different prevention and control strategy are used . The present  studies during these months include below results:

From aming the sample (1860 ) which were examined during14 months about 93 cases (5%) in honey and33 cases (1.7%) in larvae , were polluted by P.bacillous larvae.

In the apiaries which American foul brood was identified,we have done some hygienic care, prophylaxis treatment by drugs such as oxytetracyclin.

It is necessary to explain that this drug which is used for removing the disease in polluted beehive is effective on the vegetative form, and suppress signs of the disease without eradicating where as it have any effects on the spore.

Some of the infected colonies were destroyed  by burning. The bees were killed, and together with the combs were burned in a deep pit.

In the apiaries in which American foul brood was identified,hygienic,preventive and antibiotic treatment   measures were taken .When the disease was identified and the beekeepers were informed, antibiotic treatment began.oxytetracyclin was administered to the infected colonies.(OTC ) is effective in the vegetative form of the bacillus larvae . It masks the the signs of the disease.It dosen’t have any effect on the spors.And does not destroy the organism that causes the disease. For this reason ,honey infection is more than larvae infection

Discution:       `                                                                    

American foul brood is one of the most destructive brood diseases.Throug out the world,In different countries,  different prevention and control strategies are used. This study which was carried out in had the following results:

From among the samples which were examined during 14 months, 93  cases(5%) of honey and 33 cases(1.7%) of larvae had infection symptoms. In the apiaries in which American foul brood was identified ,hygienic,preventive and antibiotic treatment measures were taken.When the disease was identified and the beekeepers were informed, antibiotic treatment began. Oxytetracyclin was administered to the infected colonies.(OTC ) is effective in the vegetative form of the bacillus larvae . It masks the the signs of the disease.and dosen’t have any effect on the spores. Therefore the prevention is more important and more effective than treatment.

10 RULES FOR FOUL BROOD CONTROL

1. Make sure you are familiar with the signs andcauses of foul brood and other brooddisorders.

2. Inspect your colonies every spring and autumn, specifically to check for brooddisease. If you are unsure, seek expert advice.

3. Never transfer combs between colonies, ordivide colonies, without first checking for signs of brood disease.

4. Never bring colonies, combs or beekeeping equipment into the apiary unless you are sure that they come from a disease-free source.

5. Never buy old combs. Always sterilisesecond-hand hives by thoroughly scorchingwith a blow lamp before use.

6. Control robbing in the apiary. Never leavecombs or honey exposed to robbing bees.Never feed honey from another source to your