Good afternoon, ladies and gentlemen, and, and welcome to this webinar from the British Bee Veterinary Association. My name is John Hill and I'm president of the British Bee Veterinary Association, which was set up to really help the profession to become more familiar, say, with bees, honey bees, and all species of bees. And today, I want to talk to you about a very serious disease of, of honey bees, and it's called American foul brood.

And, one would counted it as bacterial disease and one would count it as probably one of the most serious diseases that they are afflicted with. Now, on this particular slide. We're really can just see what the honeybee is, and I'm going to go through a little bit of basic sort of honeybee natural history to start with.

So the photograph on the left shows a frame that the beekeeper would have in a in a hive, and it really shows the bees covering over what is called covered brood. And this is where the worker bees, in fact, are metamorphosing into workers. And any bee here would be very proud of a slide like that.

The photograph on the right shows a honey bee, taking some nectar from this flower, and indeed then she's also carrying out and carrying some pollen and the pollen is this little orange bundle really on her hind leg. And again we could see some honey bees moving in towards their hive with their hind legs covered or was full of pollen. Pollen is their protein source.

This is really a modern beehive, which I could show you really. It starts at the bottom of the stand, and then the floor, the floor as an entrance where the bees go in and out. And above that is a, a slightly deeper box it's called the brood box.

And this is the box where the queen lives and where she lays all her eggs. You can see a little yellow line here, which actually is what is. Called a queen excluder, and it stops the queen from going into the boxes above, and they're known as supers, and this is really where the bees, they bring in their nectar and where they in fact make a lot of honey.

And this is the the boxes that the beekeeper is able to harvest the honey from. And again you can see on the top of these frames that go into these boxes, and this is really where the what the bees use to put honey onto. They draw out these wax foundations which we give to them as a start to draw into cells, and this is where they deposit their, the honey and ripen the honey.

Now, on this particular, again, is a slightly just a cross section again of, of these particular of a beehive. So this is a double brood, there's two brood boxes here at the bottom. And then you can see this grill, which is called the Queen excluder, and this prevents the Queen from going into the upper boxes.

She's slightly too big to fit through, but the workers can move through it quite easily. And say in any normal hive, say in the midsummer, they will have about 60,000 workers or thereabouts, 60,000 workers, an awful lot of bees, but there's only one queen. And then during the summer, it will, they will produce anything upwards of about up to 2000 drones, which are the male bees.

However, in the winter time, the population reduces from 60,000 down to roughly 10,000 workers or less. There's still only the one queen, but the workers throw out the drones to die in the autumn time, so it's not a fun life being a drone. If you go on to the the occupants of the hive, the queen is really the centre of egg laying.

She is the reproductive centre of everything, and she has the the beekeepers with this little green mark onto her so she could more easily be identified if he's doing any manipulations. And there's a retinue of bees around her looking after her, feeding her, cleaning her, and helping her. Because she does, is very busy.

She can live anything up to 3 to 5 years, so most beekeepers will in fact change the queen after about 2 years. She will be at the height of the season, will be laying upwards of 2000 eggs a day, which is more than her body weight, so she is really like an egg laying machine. And she also produces this stuff quaintly called queen substance.

It is a pheromone that she produces that every bee, in the colony, all 60,000 of them will want to know to have that because then they know that she's there, and that, and from that pheromone she does tend to control the reproduction in the whole hive. And it also means is that it keeps the whole, colony cohesive. They all respond to that, to that, pheromone or smell.

She is looked after by the workers. Now certainly if there was any other queen appeared, which it won't do unless it's a a virgin, then they will all queens will, if, if I introduced a queen into a hive, they would immediately fight to the death until only one survived. There's only one queen in the hive.

Now, the workers, they do most of the work within the hive, and certainly during the summertime they will live anything up to 6 weeks, but during the winter they do last a bit longer because they're not foraging as much, and they will last anything up to 6 months. They do, the, the winter and the summer bees do have different, physiologies, but we'll not get into that. The workers do all the work inside and outside the hive.

They have what we call age-related duties, so as they as they get older, so as soon as they emerge from from their cell, they go through various stages as as their hormones or as their glands develop, their hormones change, they have different duties. However, the pheromone that the queen produces, the queen substance, in fact, means that their ovaries, the workers will not develop. That pheromone suppresses their ovary development.

Now the drones or the males, they appear by May time. They're very strong flyers and they have very good eyesight, and their main purpose in life, they don't do any work in the hive, maybe apart from maybe helping with the temperature regulation, but other than that, their main purpose in life, in fact, is to mate with a virgin queen. And really it comes to it, very few of them end up doing that, but that is their main purpose.

They live for up to about 50 days, and as I say in September or so, they're thrown out of the house to die. They've got much longer antennae, much more sensory organs, so as they can spot a queen in flight, because mating takes place on the wing. They unfortunately, if they are lucky enough to mate with the queen, unfortunately in the process of the of mating, they will in fact die after mating.

So, it is not a great reward in fact, but they die happy. They learn to beg food from the other workers, they're fed by the workers until they're thrown out. The only thing that they can do is that is that there are guard bees at the entrance to all colonies, and if any strange bee comes into the colony, they'll be thrown out by the guard bees.

Now this does not apply to drones particularly, they will be accepted in every other hive, and that is called drifting. And it it's, it is one of those things that unfortunately it has repercussions to do with disease spread. Unfortunately, the drones could be responsible for spreading disease from colony to colony.

This is really just to show really just the eggs, they are really quite small, and as I say, they will hatch after 3 days for both the the work of the queen and the drone. And they develop into larva, and these are worker larvae, and they are fed really initially for 3 days they're fed royal jelly, which is a highly nutritious food. And then they then move on to a slightly lesser quality food called brood food, and they will be looked at, but they are, these are very healthy looking larvae.

And this is really just to show what the larva development is like for the for the three casts. So the queen lays the egg, it hatches up to 3 days into larva. This is for the worker, and really at day 9, as the larvae develops, it is capped over with wax.

And then in that the, the, the larvae develops into a pupa and goes through metamorphosis until it emerges as a fully formed new worker at day 21 from the time of of the egg was laid. Now the same process occurs for the drone, except that the drone cycle lasts about 3 days longer. OK, but then the queen is a much quicker development and it's done in a special cell called a queen cell, which looks like a monkey gnat and hangs vertically.

The one thing I'd say in this diagram, the both the cells for the worker and the drone are actually horizontal, but they've been made sort of upright in for this particular diagram. But the queen is in the cell that hangs downwards like a monkey nut, and she will actually develop much more quickly because she's on this very, very highly nutritious food called royal jelly. And she would emerge as a fully formed queen in 16 days.

Now the egg that was laid at the start is exactly the same egg. That that it was late for the worker. So why then does a worker develop into a worker and why does a queen develop into a queen?

And the reason is feeding the royal jelly itself changes the hormones within the larva and the pupa, and that makes the changes the hormones so as the that particular larva will become a queen. Again, this shows the division of labour, so as when they, when the worker emerges, the first few days, they clean cells and they feed the older brood. They get the food from one of the other bees and they feed the older brood.

At 3 to 9 days they're, they're little glands within their their head, in fact, aren't able to produce food themselves, and so they're able to feed the younger brood. And that's really what happened. This is a great relevance to American fobr disease, and various, I think through various other divisions of labour, times to be processing honey, changing nectar, which is very watery into honey, which is a lot less water.

And then wax glands will be developed and they can produce wax, and then they go through periods of high ventilation and and temperature regulation. And very often at the end towards the end of their time in the hive, which is usually around about 3 weeks. They will do a bit of guard duty.

After that, as they only lived about 6 weeks, they then spend about 3 weeks outside collecting nectar, pollen, water, and this substance called propolis. Propolis is bee glue. It is It is made from resin they gather from the buds of trees in this country it's mostly poplar trees and sometimes pine trees, and they use it in fact to to make sure the whole colony is sealed against the weather, but also it is incredibly important for sterility of the inside of the colony to reduce disease.

Now, what is American fore? Well, it was named American, it's weird because it's first described in America in 1885, but it really is a global disease, global in distribution. It's been around for a long time.

It was initially described by Aristotle. And it is caused by a rod-shaped bacteria called Pinnibacillus larva. And it exists both in the spore and the vegetative form.

These spores are highly resistant to heat, freezing, desiccation, chemicals, and humidity, very, very, resistant to everything. They're also, antibiotics have really very little effect against the spore form. Antibiotics will, at the point when they hatch into the vegetative form, the antibiotics would, be able to affect the, the, the vegetative form of the bacteria.

Now, these spores are very resilient and they can survive really for up to 70 years. And they're spread really by the nurse bees when they are feeding the young larva. And this is really what, what happens, because they, the nurseries are feeding and constantly feeding the, the larvae, if they have any of these spores are present, then, then the, the larvae will be very susceptible.

Now the interesting thing about it is that it only affects American fibre, but it only affects larva less than 50 hours old. And the reason for that is that the gut lining, in the lower is very, very thin, and so the bacteria is able to penetrate it really quite easily, up to about 50 hours or so. After that, it thickens out into many layers and is therefore a much greater barrier and so it's much, much harder for the for the for at this point, the the vegetative bacteria to penetrate the, gut lining.

So the bacteria hatches from its spore into the in the larval gut and to its vegetative form that penetrates the gut wall into hemolymph. Hemolymph is bee blood. It's a straw coloured, fluid a bit like plasma, but the, the, the bacteria get into the hemolymph, they start to reproduce very, very rapidly and effectively cause septicemia.

So it multiplies very rapidly and it kills the larva, and usually by this time, the cell would be about day 9 and it will kill the larvae soon after the cell is capped. OK. At that point, the larvae starts to disintegrate and it goes into a reddish brown mush and this is the stage when we can do a test called the rope test, .

And that would get a positive road test at that stage. We'll come to that a little later. Then this red brown mush then dries out in the cell into a black scale that adheres very strongly to the sides of the wall of the of the of the cell.

And indeed it is very difficult for the worker bees to clean the cell out and to remove it. And this, this scale can contain anything over 2 billion spores, huge number of spores, even in just in one single cell. An American fibroid is a notifiable disease.

If a beekeeper suspected, he must notify the authorities and the bee inspectorates or whatever, to, to say that he suspected it's there. This is also true for almost the sister bacteria. Disease called European fibroid.

Again, a worldwide distribution of an infection, but, first described in Europe and therefore called European fibroid. American fibroid bacillus looks like this. It comes almost like little chains.

It's bacillus, they, they form into little short chains, and it's very characteristic of them if we were looking for, or to diagnose it under the microscope. Now this is a frame of very healthy brood, and if I could explain, say, is the central part here is the covered capped brood, and it looks, the cappings look like digestive biscuit colour, and this is very healthy looking. Each of these cappings.

Is slightly convex, nice biscuit colour, dry and looking very, very healthy indeed. Now just to the edge of the covered brood, you will then have a layer where you will have all the eggs and the larvae developing that will eventually become capped in a number of days' time, and the, the open cells here are really cells where a bee has emerged and it's an empty. The the workers will come in, clean it out, and then the queen will come round and relay another egg in it for the the next round of workers to start.

Then outside of the layer of larva and eggs, you will have then a layer of pollen. Pollen will be kept in this in the in these cells on towards the outside, and then this whitecapped material here, this is honey. So you have the pollen and you have the honey, so it's all the food is there for the bees.

Now, AFP symptoms, what are they like? As I said, you get the transmission of spores through the nurse bees into naive larva, and they ingest them, and then the germination of the spores in the larval guts, and then really say the you get the well the proliferation of the penicillus larvae in the mid lumen, and then these then will breach the epithelium of the larva gut and get into the hemo or the hemolymph of the larva and multiply rapidly and cause septicemia and cause the larva to die. So the larva then starts to disintegrate and use the time just after capping.

And as I say, the AFB infections very often you open a hive and you will get this sort of horrible, decaying sulphurous smell, and that's just very often where the word fobroo comes from and where people would have described this infection as being foul brood. Now they would think say oh the smell is caused by the Pennybacillus larva, in actual fact it's not. There are a whole lot of other secondary bacteria at this stage, and very often that is the secondary bacteria that cause the smell.

Now, this is really what AFP looks like. If we look at the photograph on the right, you can see instead of the nice biscuit coloured brood, you have these cappings that are darker in colour, and they're actually sunken. So instead of being convex and now concave, they're slightly greasy looking, not very healthy looking, and indeed you would look if you look here to this one down on the, on the lower left, .

Cell, you can see it's perforated. And very often it's perforated because the bees have had to look inside and they think that there's something wrong. So a lot of these cells will in fact be infected.

And the other look upon is you can see there's a lot of spaces in this particular photograph. And so the, the actual frame itself looks as if it's got what we describe as being pepper pot. Lots of, of potential holes, and really a lot of these cells will be infected.

And then what you actually get to, you could see some are looking a bit, you get this black scale. So if we go to the left hand photograph, you could see a lot of cells with this black adherent scale at the bottom of the cell that the bees find very difficult to remove. So the queen will not lay in these cells because they're dirty.

And and so eventually you get more and more cells that are empty because the queen has not been able to lay in them again. And so as I say, the disease spreads and each one of those infected cells, as I say, 2 to 2.4 billion spores of painnabacillus larva.

Now, how shall I say, the comma is pepper pot appearance, capping sunken and different in colour, may have perforations, and then the larva disintegrates into this reddish brown mucus. And this is where we do this test, this rope test. Take a matchstick or another you push it into the cell, stir it around, and as you draw it out, you get this rope of this gun, as it were, sticking to the matchstick, OK?

And it'll draw out maybe a centimetre, a couple of centimetres, and we call that the rope test. If you get that, that is almost pahemonic. You'd be pretty well sure that you've got American foul broo.

And really that this and as I say this further, once this dries out, it turns into the black scale. So the workers are unable to remove it, but the trouble is they contaminate their mouth parts with the spores because the the vegetative form is not here. Once the, the larva dies, the bacteria reverts back from its vegetative form and back into the spore form.

No, a little bit of history, as I say, it's been known all through the ages, in fact, as I say, even mentioned by Aristotle, but really to say, in more modern times, before World War War One, foul brood was very common in the UK and raised a major problem with beekeeping. And then there was some legislation brought in 1906, the bee disease order. And that advised a process called a ship swarm, where the beekeeper would take, say, the frames and he would shake all his bees into fresh new frames and fresh foundation.

OK. And the idea certainly was that they would tend to leave all the infected. comb behind the old frame.

Now you could say certainly we're not transferring some infection to the to the new frames, and that is absolutely true, but you're reducing, surely you're reducing the sheer amount of of pathogen going through a ship swarm, and it was really quite effective, it it did help quite a bit to reduce the infection. And indeed that became part of the normal procedure between the wars. However, by the time the Second World War came along, the instance, there were still 2000 cases a year of American fibre and probably at least, there probably wasn't really much statistics being kept, but a huge number of cases still by the the the start of World War II.

And it was at that stage that Winston Churchill, contacted the British Beekeeping Association, to see if honey production could in fact be increased and so reduce the amount of sugar imports to the United States coming across in convoys. And so this indeed, so during the war, there was a huge upsurge in beekeeping and the whole idea was he asked. He asked the BBKA what could we do to help to achieve good honey production.

So the British beekeepers said that they needed the destruction legislation for fibroid diseases. And that was the problem really is that there was a huge problem and it sounded a bit strange, you were saying, well, let's kill some bees, . To, to er improve the production and that's really what it was.

So really Churchill passed the legislation and diagnosis of AF American fibroid and European fibroid, meant that colonies had to be by law, had to be culled. And so beekeeping, as I say, did increase dramatically during the war. Also was there was an adjustment is that, beekeepers did get extra sugar ration to feed their bees.

Now really this graph is to show the incidence of American fibroid since the Second World War, and indeed it was very high, just during the war and certainly afterward, and then, at the same time the government introduced be inspectorate, so there were bee inspectors going out to try and look for it as well, and indeed the incidence of of American fibre dropped dramatically. And the war and the period afterwards. And indeed it was at that stage is that both diseases were counted really as one, but the AFP and the EFB were counted equally, you had to kill them.

And indeed as the inspectorate improved and did more and more inspectorates, the incidents in fact came down and down and down until really we come to today. And they say in England and Wales, we'll get something anywhere between 50 to 80 cases of American fibroid per year, so it's dramatically improved, no doubt about it. And the inspectors a great deal to do with that, and as time went on, then the number of inspectors did in fact reduce as well.

But it still goes on, there still is a strong be inspectorate and it's needed to find out where the AFP is because as you would know, as the incidence of disease gets less, of course, it's harder to find and but you don't let it get a grip, otherwise it will take off again. Now transmission of AFB, well, nurse bees acquire the spores in their mouth work and spread them into the larval cells and brood food, which is then ingested by the larva. The cell with the black black scale is a vast number of spores, which the nurse bees try to clean, and therefore infect their mouth ports all the more and it spread to more cells.

And they can then certainly it can spread to other hives, through drifting of workers and drones. Occasionally if you've, a, a weaker colony, the guard bees aren't as vigilant and therefore workers can go in to other other hives as well. Also beekeepers selling infected colonies, they may be totally unaware there's an infection there.

It is a factor unfortunately the inspectors, the ones who pick up 95% of the cases, and the reason being is, unfortunately beekeepers are, are not as good at spotting the signs and symptoms of of fibroid diseases. A beekeeper removing infected frames between hives or infected colonies to other apes. So very often the beekeeper can be responsible for the spread.

In a collapsing colonies, if a colony is going through a crisis, it's not going to survive. What may happen is that the bees that are left may abscond and try to get into other hives, in which case they could take the infection with them. The other thing is that unfortunately there's a habit of some beekeepers to feed supermarket honey to bees.

Bad idea, because an awful lot of honey sold in supermarkets has American fibroid spores in the honey. Doesn't do humans any harm, but unfortunately it would spread the disease. Diagnosis.

Well, it would be certainly the idea would be to look at the frames, the cells and see what it's like. Has it got this pepper pot appearance, has it got a smell? Are the cappings sort of darker in colour?

Are they are are the cappings sunken, greasy looking, so on and so forth. And then the, the rope test as I described earlier, if you've got a a suspect cell, that's what you would do. Analysts say it's almost pathhomonic sign and therefore highly indictive of AFP, so it is a notifiable disease, so if if the BK was suspicious.

Then he or she must contact the bee inspector and report it. And the bee inspector will make a detailed inspection and take samples for lab testing. And the inspector and beekeeper though, can use, it is a, a test kit.

It's a lateral flow test, and, you could do that for your own peace of mind. However, it is the bee inspector that makes the formal diagnosis. So this is really that the, the test gets a lateral flow, you take what you think some infected material, you mix these various solutions and then put it onto a little quite literally it's a lateral flow test and that can show you indicate whether it's positive or negative.

Right, so if you've got a positive diagnosis of AFP, what do you do? So. Once the bee inspector makes a positive diagnosis, then the apy and all hives are grounded.

No hives or bees can move out of the ay, and it is a standstill for preferably 2 inspections up to 6 weeks apart. Positive colony must be cold, no exceptions, it must be cold. And the way, what we do to do to do that is you've got to wait until the evening until all the bees have returned to the colony for that night.

You block off the entrance and then you go in through the roof into what we call the the the board at the top is the crown born, and you pour in about 250 mLs of petrol or diesel into the hole of the crown board and close it up. And the bees will die very quickly in about 10 minutes. And then all contents of the hive are put into a freshly 3 ft deep hole and set on fire.

And the next day then the hole is filled, and the reason for that is you just can't do it on the surface, burn it on the surface, because you could potentially get a pool of wax and honey that doesn't get burned properly, and then that would be wide open to to bees rubbing it again. And so the best thing to do is to fill the hole in. It's covered in soil, it's therefore out of the way of any bees robbing it.

Now, brood boxes can in fact, and superboxes can be retained and scorched with a blowtorch to kill the spores. Or you could just decide, right, there's there's a lot of infection in there, it will just burn the whole lot. Like that, so as I say the next day they this hole will be filled in.

Antibiotic use. Now, North America took the route of treating AFP and EFP with antibiotics on a biannual prophylactic routine. Different states it's, some, the auto inspected to be more stringent and others were less so.

They're just alive, had this thing, and the antibiotics were freely available over the counter. So an oxytocycline was the main antibiotic used for many years. It was readily available over the counter.

However, resistance did develop as a result, and then the beekeepers moved on to Lincomycin Titusin. And again, also resistance has in fact been reported. And the other main particular problem with this was contamination of the honey with antibiotics is a constant problem that had to be kept under control.

However, the US law changed in 2017, and US beekeepers, beekeepers must now have a vet's prescription or a veterinary feed directive to acquire antibiotics. Now unfortunately still this prophylactic use is still going on, but it is getting less and less. It's the idea is to make it more difficult for beekeepers to get hold of antibiotics, no longer available over the counter, but and I think that's the way that they should go, because really, The antibiotics will only hit AFB during the vegetative time that the bacteria is present and that really is for a comparatively short period of time.

And so really the, the antibiotics have no effect whatsoever against the spores. The UK and Europe have always universally applied a cult policy to AFP, and this has been successful and also depends how rigorously the law is implemented. Unfortunately in some European countries, illegal antibiotic use is commonplace, I'm afraid.

However, in the 1960s, UK relaxed the rules on European fog br and allowed antibiotic use. And unfortunately, this was a bad decision, and really the incident disinfection has increased considerably since. It is also a much harder disease to spot and also a much harder disease to eradicate.

So unfortunately, that was a poor decision in the 1960s. This is really just to show is really the countries that have an official policy of culling AFB. It's only in this left hand column here you can see going down, yes, yes, yes, yes, all the countries that it is compulsory to cull AFB diagnosed colonies.

Now propolis, as I mentioned earlier, propolis is a a product of where they take resin from the buds of trees, mix it with beeswax and form this material called propolis. Propolis is, bees don't change it in any way. It's really the immune system of plants that they're bringing in to the, the hive.

And they use it to block up any little areas of cracks or whatever that are very narrow. They also use it as is here in this entrance of this nest, to be a bit like a foot bath coming in because prolisis is great antibactmicrobial properties, causes bacteria, fungi, viruses, everything. And and so it means that the inside of the hive, which the bees coat in propolis, this is the inside of a hollow tree, they coat it with propolis and it stops any further rotting straight away, but also provides a very, very clean, sterile environment within the colony, which is fine when you've got anything up to.

You know, 2030, 40,000 members in a in a colony, disease could break out very, very easily, and pros is there to stop it. Now, so this is really just to indicate that propolis will have an effect at reducing American fibroid disease. And so if we take the the pader columns here, there's no propolis, and then they introduced bacillus larva, and indeed there's less .

Or be die from AFP. And so, and that does really seem to be that and right through it, it does show that it does have an effect. So problems is useful from that point of view, but it will not eradicate the infection.

But it is part of this thing we call social immunity in a bee colony, which is very, very important for not only against fibre but all other diseases as well. So the problems in honey bee colonies plays a more subtle role in colony level immunity. So the level of pathogens, say for example, American firebird also cholkri is a fungal disease, it's reduced, greatly reduced chore is a very common disease, and it's a fungus that's there a lot of the time, but the, but the problems will reduce that.

But it means then really, you know, the problems is able to help that, it means that the bee does not have to generate as as many. does not have to generate as much immune response, and so it's, it's less, stressful on the colony and it's under less pressure. So that, that then means that the overall system, the bees can concentrate on other things, there's less energy wasted on immune defence.

Now we're coming really to what the is really an incredible new development really in in the whole story of American fire bred. Now this is, researchers, it was led by Professor Dalia Freitag in Helsinki University, and she has moved now to Gras University in Austria, and she has successfully vaccinated bees against American fire brood. And this is the first ever insect vaccine.

Using the process of transgenerational immune priming. OK, she formed a company called Dalan in Los Angeles, and it was to progress work towards applying for a veterinary licence in the US, and a condition licence was in fact achieved in 2022, and they're doing further work to progress on the efficacy and really say it is a thing that they're pushing forward to see indeed if they can get a European licence and so possibly one in the UK as well. And it's also the fact is whether this transgeneration immune priming can be applied against other bee diseases such as European firo, chalkbrood, and the bee viruses, which are a major problem at the present time.

And really say an extension of this could very well be if this all works, that we're coming on to insect farming for protein production, say for, for animal feed or even human feed. Is that, is that being able to vaccinate, insects against various diseases will be immensely useful, in the future of protein production, insect farming for protein production. Now, to go into just what is bee immunity, everybody used to think that the bees had a very primitive immune system.

I always thought that was a very dangerous thing to say. Bees have been around for 34 million years in their present form as as these social insects, and they've learned a thing or two. I mean we've only been around for what, 5 or 6 million years.

The bees have figured out a lot of things in the meantime. So you take the individual bee. They're the cuticle, so they've got an exoskele which keeps out infection.

The saliva has enzymes in it, which will help to reduce infection. They groom each other greatly, and also the symbiotics are microbiome, although, not so many numbers of bacterial families very, very effective. And it's really is a very good set neutralising along with this sort of thing to keep the disease agents down.

And so their own immune system, they don't have anti antibodies, they don't have a lot of specialised cells. They do have a few, some white cells, but they're not in huge numbers. But what their own immune system does produce is they produce antimicrobial peptides, chemicals.

They have a little thing called RNA interference, which is quite common in the insect world, in which the the the RNA in fact is changed, so as it becomes the organism that's trying to move into is no longer able to function. Now that is not genetic engineering, this is a natural RNA interference is a natural process within the animal world. And then with these antimicrobial peptides, you've got things like foetal foetal, oxidase, and, it is one that apparently we have in our systems as well.

Also the, the few cells they do have in their hemolymph, they can undergo yocytosis or indeed encapsulate pathogens. Now if you go on to a colony, a principle, then you've got propolis, which I've explained to you before, is very, very antimicrobial. They undergo hygienic behaviour in which they will always say if there's a path that they will try and take out diseased larva, diseased pupa, get rid of them, and I would call that hygienic behaviour, very common in a beehive.

They also will sterilise food both by enzymes that they will add to the food and also say for say the sterilisation of pollen, bringing in pollen so it doesn't go off, they will put into a cell. Add some honey and allow it to ferment, so it reduces the pH and so the protein will be preserved for much longer periods of time. You get this task specialisation of nurse foragers, so the foragers out foraging, whenever they come back, they hand the produce that they've gathered, they don't take it directly into cells, they hand it to receiver bees and nurse bees in the colony.

So in fact there's less chance the foragers are bringing disease in they hand it to the nurses and so there's less traffic, as it were, going through the colony. They're very good at if, if a bee dies, they will get it out of the hive and lift it out the hive very, very quickly. And then the social ability principle I talked about before is thermal regulation.

A beehive, if you put a thermometer in a beehive, any time of the day or night, it will be at 35 degrees. Centigrade. It is exactly kept at that because that's the ultimate temperature for a root to develop, in the cells, and it's kept at that morning than at night from really from the early spring right through to the late autumn, when they do take a couple of periods of a couple of months at a slightly lower temperature, but the rest of the time the hive will be at 35 °C.

If they think there's a lot of pathogens around, they will raise the temperature in that area to kill it, by beating their wings and raising the temperature much higher than 35. There was the sharing of these antimicrobial peptides, and as I say, the order of hygienic brood removal, any dead or infected larvae, they will try and get rid of them. So that is the what what actually goes on in their natural history.

Now, if we take, say, just the queen here, and we give her some pathogens, be it say bacteria or a fungus or a virus, she will assimilate that in in her gut and it goes on to this thing of a toil receptor, and that is really that she will generate or to be able to turn on various genes. That will be specific or general, and the idea this is done in fat body cells. Now, this is not adipose tissue.

Fat body is a name given to any of the white tissue you see in a, in a larva or a pupa. It's not fat adipose tissue, it is actually liver. Very, very important tissue indeed.

So, so the, the, the, the queen, said the queen in this case will recognise so activates the the specific genes. The gene expression takes over and produces, say, antimicrobial peptides, such as these ones here. And then it goes on and to encouraging encapsulation, which is a little bit like phagocytosis, optimisation, which in which the chemicals will be produced, which will adhere to pathogens and make them, much more likely to be phagocytos, and you get the celllysis and and so forth.

So, as these things go on, that would normally would be what would happen. Now with transgenerational immune priming. The, again, the same thing will happen with these particular receptors.

You will get, say, specific. Gene expression to go on to produce antimicrobial peptides and generally to have to push on for these other things like phagocytosis and capsulation, so forth, but also say these are produced, but they will in the queen go into what is called the telegena. Now vitallogenin is a remarkable chemical which we have, it's in every egg, in mammals, in birds, and everywhere there is vitallogenum and vitallogenum acts like a transport medium and makes up a huge amount of the yolk that goes into eggs that is there to feed the the developing embryo.

And what will happen is that these antimicrobial peptides, once the the queen has been vaccinated, which we will come to shortly, then these peptides will go into the telegenin, they then go into the ovaries of the bee, and they will then be then moved, the, the chemicals cells will move into the eggs, which then develop into the larva and the actual larvae then will be vaccinated against that particular pathogen. This is just a thing of what the talagege looks like, very complex but very important chemical. Now, vaccination.

So we're what we're talking about here, say is Bactrim, that really is mushed up AFB suspension, OK? It's called bacterin, and the, the process of that, in fact, is it is at the moment confidential. The idea certainly is that the bacteria is mashed into pieces and is then mixed with sugar.

It's put into a little cage like this, and there were some worker bees in here, and they in actual fact, will take it in the vaccines and they will incorporate it into royal jelly. And then these workers feed this vaccine to the queen. And then the queen bee, the bacterium is absorbed into the fat body cells and then transferred into the ovaries.

OK. So the idea then is that the components of the bacter bacteria or the bacter I should say, stimulates non-specific immunity in the larva, and the bacter bactrin is transferred to the eggs and hence the larva in the hive. So, this queen or sugar syrup or candy is composed of dry sugar and corn syrup in a in a ratio, and then the bactri is added and and so then fed to the workers who then feed the queen.

So, let's to say then the the young larva was susceptible to this disease for a very short period of time, . After hatching, but after this time, they're resistant to AFB infection. So the immunity may work through the toll receptor pathway or other receptors, resulting in the production of antimicrobial peptides or non-specific cellular immune mechanisms such as optimisation and direct lysis.

So that is the way it's the vitalagegenin helps to transport this and this in fact helps the the larvae to become immune. So, I'll not go through this too much, but the whole idea they had to like go through various trials to to understand to get to the efficacy. So the queen bees are given the oral vaccine via the queen pages, so there's control over the vaccination procedure and the duration of exposure to the androgen and exposure to environmental factors.

So queens are introduced to the hives, and then once they're accepting the hives, she lays the eggs immediately and the hives are assessed to ensure there's no adverse effect of vaccination. And also for Queen stance disease status. So a challenge to ARP can't be done situs, the larvae are only to substances for up to 36 hours after hatching.

So after about 2 weeks, the larva taken 24 hours after hatching exposed to high doses of pendbacia larva, OK. And then really what we're tend to be is that typically 20% of houses might be affected by other outside factors, and that really has to be taken into account. So, so, Pataciarvis is spore forming bacteria.

Other spore-form bacteria could be the Clostridia anthracin produces anthrax or Clostridium teta tetanide which produces tetanus. The AFB sores are subjected to a process which breaks them up. They must be rendered non-pathogenic.

The process is commercially sensitive sensitive, and so I, I can't describe it to you. This is really just to prove is that these antimicrobial chemicals actually get through to where they're required. And these have gone through a fluorescent process, and so they're ingested, so they're actually in the midgut here, and then in the next time they moved on, they've moved into the fat bodies, which is, I think it's sort of white and .

In the bee and and it's affected this equivalent of the liver, and then in fact into the hypopharyngeal glands, and these are the glands in the head that produce brood food. So these chemicals are in the brood food which the which the worker bees are then feeding to the queen. And then the last thing is that they're seen in the queen, in the ovaries in the area where the eggs are being laid.

So that to prove efficacy, as I say, the, the queen bee is immunised the labs and the kill pathogen that was added to the feed is monitored vitality for 7 days, transferred to new hives, she starts laying eggs, and the researchers then collect frames containing 1 day or larva and take them to the lab. And they're then put into petri dishes and they're challenged with American fibrid bacilli. And indeed then from that they they're either going to be alive, they're going to be dead, so it's either one or the other, and really from that they they were able to show in fact is that when they were AFB challenged, they, the larval mortality was much less than in the the in the unvaccinated larva.

And the efficacy results as they sort of tend to, to get to, and the initial trials, they got 30% efficacy, and in the further trials in Spain, they got 54%. Now you say, oh well, that's not particularly high, but the, the one thing I would say is that when they were doing these trials, whenever they were, in fact, subjecting the larva to the AFB bacillie. They were doing it in vast quantity, much, much higher than you ever would have experienced in the field.

And so the idea was really to see if they could be still robust and indeed they were still getting at one point 54%. I guess a very, very, very high challenge. So it, it is certainly means that there is scope here that there that this can be improved and that indeed that we will have a very good viable vaccine for American fibro.

So in conclusion, really American fibroid is a global disease. It is ubiquitous spore forming bacteria that can survive up to 70 years. Highly infectious, and beekeepers must keep vigilant observation to identify infected colonies.

Antibiotics often only mask the symptoms and can adversely affect the bee's microbiome. That's another little problem really is that it does have significant effects on the normal bee microbiome. Prompt destruction of infected colonies and biosecurity measure key, and the development of an infective vaccine offers a new weapon against this disease globally.

So I really would just like to thank Professor Feritach for her for her allowing me to use some of her slides and also for her help. Professor Budge at the University of Newcastle, who is, I think one of the UK's leading authorities on fire bridge, Professor Maurice Sivak from the University of Minneapolis, and she's a world authority on Propolis, Doctor Kirsty State of the National B unit. Megan Seymour, who is a regional, be inspector, and Fight to Europe, who produced the, the lateral flow tests.

And certainly if there's any questions that you would like to ask, and certainly, I'll probably receive these by email and certainly no problem if you wish to send them, I will reply to them as as soon as I can. Thank you very much indeed.