Science Down Under 2010

Chris -   Now from the depths of deep space to the depths of your stomach, and if you've ever been afflicted by an ulcer or gastritis, here's the man who won a Nobel Prize for discovering the bacterium -  Helicobacter pylori -  that is responsible.

Barry -   I'm Barry Marshall.  Here I am at the Q.E. II Medical Centre in Perth.  It began in about 1981.  At that time, I suppose I was 30 years old, and I met up with Dr. Warren who had seen these curved bacteria on stomach biopsies - he's a pathologist you see.  And so there was a curiosity for us to investigate this further:  How come bacteria can live in the stomach which is supposedly full of acid and kills bacteria?  So nowadays, we know that bacteria can live in hot springs, but that was pretty much new stuff in 1981.  And so we set out to perhaps identify and culture these bacteria and I went off to interview all the patients that had them on the biopsy to see what was wrong with these people.

Chris -   What made you think there might be something in the clinical history that'd be worth investigating?  Why did you decide to follow that up?  Because it would've been easy to just say - oh there are some bacteria in the stomach - maybe we missed them before.

Barry -   Well, they were new so obviously there was no knowledge about them, so we had to exclude the possibility that they were causing a disease.  And when we did interviews with people we found that there were one or two symptoms that seemed to be connected in some way.  Dr. Warren knew already that they caused inflammation in the stomach - which was gastritis - but the teaching was that everybody got gastritis if they lived long enough and who knew what was caused by spicy food, or smoking, or alcohol, or some of the hundred different explanations? 

But according to his pathology results you really had to have the bacteria to have gastritis.  So we then set out to find - what about people who don't have the bacteria -  do they have white cells in the lining of their stomachs?  Do they have any symptoms?  And we went off with open minds just trying to find out what the patients would tell us, and we came back with nothing initially because people could have indigestion, or belching, or dyspepsia, or nothing, or anaemia, all kinds of weird things, or ulcers, but nothing came out in our pilot study. 

So we said - okay let's do it properly now - we'll do a prospective study.  We'll look at 100 patients, we'll take every single patient having an endoscopy down - so a very big mixture of people.  It was done blinded, so you didn't know what was wrong with them or what the results were, and when we looked at the results, the ones with ulcers had the bacteria far more commonly than just anybody else.  So that was a significant result, and that was a year later that we had that result and we had already studied the literature, and it all fitted together all of a sudden.  Hang on a minute - maybe ulcers are not caused by stress.  Maybe they're caused by a germ in the stomach!  Nobody had ever thought of that, because supposedly you couldn't actually have bacteria in the stomach because the acid kills them.  So once you say - well that was wrong - all of a sudden it's wide open.

Chris -   The question is though - it's one thing to say there are bacteria in the stomach and there's an ulcer, but how do you resolve the question - the bacteria are there because they've caused the ulcer, rather than the ulcer has made the stomach environment change in such a way that it now allows these bacteria to persist?

Barry -   I'm getting déjà vu because that's exactly what people used to say who didn't believe us.  And so we said - we have to prove that these bacteria are harmful or harmless.  And of course, that is an experiment called Cox's postulates, where you have to infect an experimental animal with the bacteria.  But sadly, we didn't have a successful experimental animal, so we had to then look for human volunteers.  And Warren and I discussed this and he couldn't be a volunteer because he'd already had Helicobacter and I had treated him, so he would've been a bad choice, and I hadn't had them. 

So it fell upon me to do the experiment.  So I did drink the bacteria - a few tablespoonfuls - not very much, and it didn't really - well actually, it's not published yet, so I won't tell you what it tasted like!  It wasn't too bad.  So then I was just waiting to see what would happen.  By then I knew that there were lots of people who didn't have ulcers who had the bacteria, so I was there like - I suppose nothing will happen because none of my ulcer patients can remember catching a bacteria, so probably they're not going to cause any problem.  So I was of course surprised when about five days later, I woke up and ran into the bathroom and started throwing up, and I was vomiting for about three days.  There were a lot of vague symptoms but nothing that you could really hang your hat on. And then after that I was okay, and the endoscopy was done.  And sure enough, I had millions of these bacteria, thousands of white cells all trying to eat the bacteria - so it was really a bacterial infection that I had in the stomach.  So that answered this question that said - healthy people with nothing wrong with them could catch this bacteria and then get inflammation in the stomach -called gastritis. So that was then the soil upon which an ulcer would form later in life, and so we connected the whole story up at that point.

Chris -   So what is the bug actually doing and why is the immune system not dealing with it?

Barry -   Well, it's a bit like D-day.  All the ships are parked offshore, out of range, firing at the coast - and so  Helicobacter is like that, living in a mucus, hammering away at the epithelial cells, making them a little bit leaky, so that they leak iron and nutrients up to the bacteria, so that's the happy status for the bacteria.  As soon as you get too many white cells migrating through the epithelium in the stomach, it's starting to develop holes in it, it becomes leaky, acid goes down, and you develop an ulcer.  So that would be disadvantageous for the bacteria.  They don't want to kill you because then they can't propagate. 

So a successful bacteria lives in sort of symbiosis.  So what they want to do is keep the inflammation going, but not so vigorously that you'd die from a bleeding ulcer.  So an ulcer is actually [when] the bacteria is a bit over zealous, if you like, in harming you, it doesn't want to do that. It has toxins, and one of these toxins called the VacA toxin probably down regulates the immune system.  So as soon as you get too many holes in the mucosa, this stuff would be leaking into the lining of the stomach and inhibiting the proliferation of the white cells.  So that's the kind of thing that it would be doing.  So your immune system then is a bit paralysed and doesn't actually become more vigorous as it would do.  So, you can imagine maybe 500,000 years ago, the first Helicobacter infects humans - it is too dangerous and the poor old humans probably die from bleeding ulcers.  However, one of the Helicobacter mutated and developed this immune suppression system and got the right balance, and now we all have Helicobacter all our lives, or we used to, but in the last 100 years it has decreased to about 25% of population in most countries. 

So you can pick it up as a child and it sits there, controlling the immune system, regulating it for the rest of your life.  And so it's a very mild irritation in the stomach, and most people don't know that they have it.  10% of people at some time in their life develop ulcers and that is a major event because ulcers then come and go.  So it would put you in the high list acid level and then if you have Helicobacter all your life, that irritation and inflammation attracts stem cells floating past and these stem cells try to fix up the problem in the stomach, trying to make it heal, and they're proliferating and they are subject to hits by carcinogens that you might eat, so you do get 1 or 2% chance of having stomach cancer if you live as long as a Japanese person - you know, 80s or 90s.

Chris -   Barry Marshall - the man who ate his own experiments to prove that the bacterium Helicobacter pylori can cause stomach inflammation, ulcers, and even cancer.  And Barry also told me that he's now working on a way to produce tamed forms of that bug, that can be used to deliver vaccines against other diseases like the flu.

Chris -   You may not have heard of a Bilby, but believe me, these mini marsupials are extremely cute.  But they're also an endangered species in Australia alongside many of the country's other native fauna, largely owing to feral animals, like cats and foxes, that have been introduced from Europe.  But now, a major initiative called project Eden which is based in the Francois Peron National Park in Shark Bay, Northwest Australia, has been set up to tackle the problem by returning a patch of the country to what it would've been like before rat and cat bearing European settlers arrived on the scene.

Brett -   I'm Brett Fitzgerald.  I'm the District Manager for the Department of Environment and Conservation in Shark Bay District.  The aims for project Eden is to reconstruct the original native fauna on the Peron Peninsula within Shark Bay.  So that's done through a combination of getting some information on what species were here originally, then managing the actual pressures that are on those species, the ones that were remaining and the artificial pressures that possibly pushed some of the others to extinction.

Chris -   And what were they?

Brett -   The main pressure that we've seen on, not only the Peron Peninsula but across Australia, is the introduction of feral predators.  The feral cat which has been around for longer than foxes, but in synchronisation with that are species such as rabbits which have provided a prey source and a food source for the introduced predators.  This lets them build up to numbers, and then what appears to have happened is we have a crash of those introduced prey species, such as the rabbits, and then the predators are at such large numbers that they move across to what are possibly remnants of smaller populations of native animals and push them towards extinction.

Chris -   So why have you chosen this site to try this experiment, to try to make this happen?

Brett -   Across a lot of Australia where we've seen native animals survive, native species survive, has been on an island environment because we haven't had the feral predators introduced.  The difficulty with working on islands is simply access.  They're remote and logistically they're difficult places to work on.  Peron Peninsula inherently was a mainland island.  It's 100, 000 hectares, so it's 1,000 square kilometres, but there's only a narrow neck, about 3.2 kilometres wide which connects that large peninsula to the mainland.  So by fencing that off, controlling feral animals inside the enclosed area, we can limit the potential for re-invasion and I guess, get the best value for money for the eradication programs.

Chris -   What have you done in terms of getting rid of those nuisance animals that us Europeans brought here a couple of hundred years ago?

Brett -   We've controlled the large herbivores.  So, species such as feral goats and sheep, and Peron before it was purchased for conservation was an operating sheep station and goats were not really used commercially in those early days, but they were present here as well.  So that's a combination of mustering, trapping and eradicating those species.  They're large.  They tend to come to water in summer, so they're relatively easy to control.  Then we had red foxes which are the common space in Europe, but introduced to Australia.  They tend to be relatively easy to control by a naturally occurring poison placed in a bait here.  So, the estimates were between 2 and 3,000 foxes on the Peron Peninsula before control commenced.  We have the occasional one that might sneak through the fence or sneak around the fence, but we're in the order of single figures of foxes in the peninsula.  The fourth major species that would be in looking and controlling is the cats.  Unfortunately, control of feral cats is an incredible challenge.  They've got a great breeding capacity.  They don't need to drink water.  They are quite adaptable with the pride they select.  So they're a difficult nut to crack in a sense as far as eradication goes and they've proved a big challenge - as have rabbits.  The rabbit population here tends to fluctuate with season.  We have periodic outbreaks of natural diseases such as myxomatosis which depletes the population, but rabbits have an incredible breeding capacity, and even when the numbers are suppressed, once we get some reasonable rainfall, they bounce back, and they're also an issue.

Chris -   Brett Fitzgerald. 

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Chris -   And apart from just eradicating the feral animals to let the plants and native species recover, project Eden also has a captive breeding program to help kick start the process.  Colleen Sims...

Colleen -   Let me show you here one of the animals that we've been breeding.  This is a Bilby or more correctly, it's called a Greater Bilby.  These guys used to be very common across Australia, around about 70% of the country.  They are a marsupial and a burrowing marsupial, so one of the unusual things about many of the burrowing marsupials is they have a pouch that points backwards, so they don't fill them up with sand when they dig.

Chris -   That could be a disadvantage, couldn't it?  It's about the size of a rabbit, isn't it?

Colleen -   That's right.

Chris -   It's like a small rabbit.

Colleen -   Yup and as you can see, the very long ears, one of the common names was Rabbit-eared Bandicoot.  The Bilby is one of the bandicoot family.  They're actually omnivores rather than a herbivore like a rabbit, so they'll eat anything from seeds, root tubers, insects.  They also have been known to eat mice and small geckos and things too, so they'll pretty much go for anything that gives them a good feed.  They have some fairly sharp teeth obviously, considering the array of food they eat.  The other thing that's a dead giveaway for their behaviour is if you look at their forearms, they've got very strong short forearms and very long claws for digging on their front feet.  They can go along on a fairly good clip.  I guess it's fairly similar to a rabbit gait in fact, a sort of a half hopping gait to start with and once it gets faster, it will actually really get running.  They are nocturnal so they live in burrows during the day, anything up to about two or three meters deep, so they can move a lot of sand and move it very fast.  They actually were a fairly common food source for the aboriginal people and still are in some of the remote desert regions, but it takes a lot of effort to actually dig a Bilby out of its burrow.

Chris -   So given they can hide underground, why are they particularly vulnerable to things like cats and foxes then?

Colleen -   Well, the reality is they probably are one of the less vulnerable.  The fact that they actually do still exist and persist in Central Australia at this stage means they're doing a lot better than many of the other species we previously had here.  That doesn't mean they're immune and there's certainly ongoing concerns that they may still be slowly shrinking in numbers.  Because they're nocturnal, because they are omnivores and move about the landscape depending on food sources, it's quite difficult to monitor them.  Often, you can return to an area where there was quite a thriving Bilby colony and several years later, they aren't there anymore, and it does create problems to work out whether those animals, that population, has actually gone extinct or whether it has moved somewhere.  But there's certainly ongoing concerns that even though they were persisting in the desert areas, that population and range may still be shrinking.

Chris -   So when you want to breed an animal like this in captivity, what does that actually involve?

Colleen -   Obviously, it initially involves getting the original animals from the wild and setting up a program.  We have a fairly intensive management program here to try and manage the distribution of the genetics through the breeding population.  These guys are actually comparably easy to breed.  They've actually been partially adopted in Australia as a replacement for the Easter bunny.  Obviously, the similarity and appearance makes them quite convenient for that, but also because they are actually quite a rapid breeder, so the fertility aspect is actually carried on.  so we have an Easter Bilby these days in Australia and will usually get anything up to three or four litters out of these guys in a season in a year.  So, they'll produce usually a litter of two, but anything from one to three.  We will usually, based on some advice from the national stud book that gives us an idea of the relationship of various Bilbies we have in our colony, will pair a male and female.  They may stay paired for most of their time here.  Once we've had three or four litters, we might actually want to move pairs about and re-manage their pairings and the genetic lines from that.

Chris -   So when a baby pops out, it first of all goes into this backwards facing pouch.  How long does it spend in there?

Colleen -   The Bilby, as I mentioned earlier is a bandicoot.  They actually have a really rapid reproductive rate.  Their gestation is only about 10 days, so as with most marsupials, the foetus comes out very small and very underdeveloped, and they then spend around about anything from 65 to about 75 days in the pouch.

Chris -   And are they sociable?  Will they hang around with a family group or do they go off on their own and act independently as soon as they get out of the pouch?

Colleen -   They'll usually spread out and disappear.  In fact, some might think there's a great idea we should do with our kids, but the Bilbies, the females actually will leave the burrow, the home burrow to the kids.  So she's the one who basically leaves them after around about two weeks after they've come out of the pouch.

Chris -   Colleen Sims, who's a vet and project officer with Project Eden. 

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Chris -   But apart from the important task of increasing the numbers of endangered species, there have also been some other surprising discoveries.  Brett Fitzgerald again...

Brett -   It's interesting to actually watch the re-introductions, it's so much more than just putting back the small furry animals.  When you actually look inside the breeding pens of the Bilbies and when you look at areas where they're being re-introduced, these actual deeper diggings that they do, you can actually say then why that functions as part of the ecosystem.  A lot of the seed that's distributed in Australia is airborne and wind driven, and you'll actually see in these deeper holes that Bilbies dig the actual seed fall into the bottom of the hole then having some dirt below over the top of them.  So as far as driving the ecosystem, as far as plants germinating and getting a chance to survive, you really see the function the animals play as much as just their presence in general.  Water penetration; when you look inside a Bilby pen or around a Bilby related area, you may have these shallower rabbit diggings, but the Bilbies really turn the ground over.  When we have a rainfall event after that, the capacity for rainfall penetration - water to actually soak into the soil rather than runoff and disappear is that much increased by the presence of these animals.  So, that's been a really interesting part of the project.  It's not just to see the animals back in their natural environment, but to actually see the ecosystem function they played.

Chris -   Do you think it's been successful though?

Brett -   I think we've had some real successes.  Some of the species such as Malleefowl, from the process of going and collecting eggs to actually hatching those eggs, to releasing those birds and then actually going in and finding active breeding mounds on the Peron Peninsula.  Some of those species, Bilbies included, we've seen real success and it has essentially made Peron a site where we can potentially take those animals bred here to other locations and really use this as a launching pad.  It's been challenging, and feral cat control is a challenge, but as well as the effect of control here, the other things we've learned is better techniques.  So, it's a learn-as-you-go process with some of the species and some of the feral control.  The techniques that we've developed here and the things we've learned about cat behaviour have been able to be applied, not just in Australia but in other parts around the world.  So, I think we've had some real success with the species re-introduced here and we've learned things that have been able to be applied in other places.

Chris -   Brett Fitzgerald from project Eden in Shark Bay Western Australia.

Boris -   Reproduction in honeybees starts very often when the colony gets fed up with the queen and they execute her, chop her into bits and pieces, and thrown out of the colony.  They then use the last eggs which she has laid before, and they start to breed new virgin queens.  And When they hatch they are normally separated for a while until everybody has hatched, and then they again fight against each other until one or very few are left.  These queens then leave the colony for what we call a mating flight and they go high up in the air, they release a pheromone, a substance that attracts males, and the males come in large numbers.  The ratio between virgin queens and males is really extraordinary in honeybees, about 16,000 males and one female.  They all queue up behind her, forming what they call a comet, so they basically queue up for copulations. The female then copulates with about 200 of them, or up to 200 of them.  She kind of basically just collects ejaculates, she goes back into the colony, and then she sorts these ejaculates out, but only very few, about 5%, of the sperm that she accumulates during her mating flight will ultimately be stored.

Chris -   So does she then use that sperm that she selects for the rest of her life because queens can live a long time, years?

Boris -   Yes.  In honeybees, they can live up to 7 years, in other social insects which we work with they can live for decades.  So we know that for example the ants which we use for comparative studies, they can live for about 20 years.  They only mate very early in their life.  Once they have laid their first egg, once they've initiated a new colony, they will never go out and mate again.  This has very dramatic consequences on the female because they have to store immense amounts of sperm, millions and millions.  They have to keep it alive for thoe whole time and they have to use it very prudently throughout their lives to fertilise eggs.  And it's quite fascinating, how can females, how can queens do this so efficiently?

Chris -   So are you any closer to understanding then how they sort out this sperm?  How do they decide of all these 100s of matings?  "I want that sperm and I'm going to use it in whatever order.  I'm going to flick out that sperm, I don't want that."  How do they do that?

Boris -   I think we started to get first insights into these.  The male doesn't only transfer sperm to the female.  The male also transfers a secretion which we call seminal fluid or seminal plasma, and that seminal fluid consists of proteins, and when we looked at these proteins, we saw that they could be involved in little warfares between the ejaculates but also between the males and the females.  And we tested that in honeybees and what we actually find is that some of these proteins seem to be able to recognise none-own sperm and kill that sperm off in the female sexual tract.  In the end, when we replicated that work, we could also study the female side of this and in these ants we found that the female secretions can actually cancel out that little warfare.  How you can imagine this happens is that you have a picture in front of you of a gladiator in an arena, the ejaculates then enter that arena which is provided by the female and it's a sexual trap that they fight against each other in.  We think the weapon here which they use are these proteins and then you have losers, you have winners, but up there, the spectators, they will be the females, and they make like in the old Rome, they make this really famous sign of thumbs or thumbs down.  So they ultimately decide who will win, who will father offspring and who will be chucked out.

Chris -   And do we understand what the nature of that biological warfare is?  What those proteins are doing and how they're recognising, "This is my sperm.  That's a competing sperm.  You're dead.  My ones are going to survive?"

Boris -   That's actually what we have just started working on.  We don't know yet what these specific proteins are or what the recognition process is.  What we tried from our list to do is to identify those proteins that are there to then test them specifically to find out whether they are responsible for that effect or not.  Initially, it sounds weird that a liquid can recognise own from non-own or a simple cell like sperm cell will be able to do this, but there are a couple of studies that have been published over the recent months actually, that all indicate that it's actually something that should be there.  There was a similar study that was performed in mice a couple of weeks ago.  It was found that sperm in mice is also able to recognise own sperm from non-own sperm.  So they basically align themselves to kind of cluster together, but only those from the same male will form that alignment.

Chris -   I'm understanding how this works.  Presumably, this is going to inform better bee conservation and bee breeding strategies.

Boris -   Yes.  That's one of the ideas which we have that deeper knowledge about bee breeding, bee reproduction will help the bee industry to kind of breed better bees and to kind of ship semen samples around for artificial insemination programs, and we have an artificial insemination lab here so we're actually involved in-breeding of more bees or bees of better quality.

Chris -   So you're effectively creating a bee sperm bank.

Boris -   Yes, to a certain degree.  The advantage of the bee sperm is that you can keep it in a glass vial for a very long time.  It survives for a couple of months without help and at room temperature.  So, if you need a lineage which has an increased tollerance or resistance against a parasite, and that's at the moment a big issue, then you can - instead of importing bees which is always risky - you can import semen.  For some of the more dangerous diseases that travel around the world in the bees like the Varroa mite or the hive beetle we know that's not present in semen.  So we have a lower risk of importing something which we don't want to have here.

Chris -   Bee Biologist, Boris Baer - he's from the University of Western Australia.