Una Ryan is an Associate Professor in Biochemistry at Murdoch University in Western Australia, where she also received her PhD. She obtained her BSc degree from University College Dublin in 1988 and has been working at Murdoch University since 1989. Her research interests include understanding how pathogens are transmitted and developing novel therapeutics for pathogens. She was the recipient of the 2000 Science Minister's Prize for Life Scientist of the Year. She has published more than one hundred scientific papers and obtained more than $1.7 million in research funding since completing her PhD in 1996.

Combating parasites using novel technologies
by Associate Professor Una Ryan

I am going to start by giving a brief overview of parasites, and then talk in particular about the parasite I work on, which is Cryptosporidium.

It has been estimated that the volume of water going over the Victoria Falls every day is roughly equivalent to the amount of diarrhoea that is produced every day as a result of enteric parasites. Parasites have an enormous influence on our lives. They impose an enormous health and financial burden. They are extraordinarily diverse – there are actually more parasitic organisms than non-parasitic organisms, and they represent more than 50 per cent of the world's biodiversity.

We are still a long way from overcoming parasites. There are few effective vaccines, and one of the biggest problems we are facing is that many parasites are developing resistance to various drugs, and many parasites that we thought we had dealt with in the past are now undergoing a resurgence as a result of this.

Parasites have an enormous impact on humans. Humans can suffer from a wide range of diseases such as sleeping sickness, leishmaniasis, filariasis et cetera, all of which can be fatal, and then there are ectoparasites: scabies, lice, fleas and mosquitoes,which  can be very important in transmitting these diseases. There are over 300 diseases infecting humans and they are responsible for about 80 per cent of deaths in developing countries, so they represent an enormous burden on the human population.

Guinea worm
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This is guinea worm, which is endemic in many parts of the world. It is transmitted by infected water, and if you drink the infected water the parasite undergoes its life cycle and then the female worm erupts out through the skin like this. Sometimes they can be over a metre in length. You have to actually wind it around a stick to get the entire parasite out, because if it breaks off while it is coming out it actually releases more infective stages – perpetuating the infection.

Filariasis (elephantiasis)
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Filariasis is often referred to as elephantiasis. It is transmitted by lymphatic filarial parasites, and it is estimated that 1 billion (20% of the world's population) are at risk of acquiring infection, with over 120 million infected worldwide. Ninety percent of these infections are caused by Wuchereria bancrofti, and most of the remainder by Brugia malayi.. One of the clinical manifestations is this massive lymphoedema here [in leg] as part of the infection, and tragically once it gets to this stage there is absolutely nothing you can do about it.

Malaria
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We used to have malaria here in Australia. It was eradicated in about the 1960s, but unfortunately malaria around the world is seeing a big resurgence, largely due to an increase in drug resistance.

This [at top left of slide] is an uninfected red blood cell; this [at top right of slide] is a red blood cell that has been infected with the malaria parasite Plasmodium. As you can see, it totally changes the red blood cell. One of the effects is to make the red blood cell sticky and adhere to other red blood cells [as shown at bottom of slide] and the small vessels in the brain severely restricting the flow of essential nutrients to the brain.. This is responsible for some of the clinical effects that you see.

About 2.3 billion people, about one-third of the world's population, are at risk of infection. Sadly, every three seconds a child dies of malaria. And even though it has been eradicated here in Australia, a lot of people are coming into the country with malaria from overseas. Many of our troops who went to East Timor, despite the fact that they had been given anti-malarials, came back with malaria. And so it is a very serious threat for the future.

Trypanosomes
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This [on slide] is a red blood cell, and this is a trypanosome. Trypanosomes are responsible for an enormous amount of death worldwide. Sleeping sickness is caused by T. brucei, it is almost always fatal and about half a million people are infected, with 55 million people at risk.

Another species of trypanosome (T. cruzi) causes Chagas' disease, and another species (T. evanesi) causes a disease referred to as Surra, which is not in Australia but is endemic in some of our closest neighbours – Indonesia and possibly Papua New Guinea. It infects a huge range of hosts and results in mortality and reduced productivity. We know from experimental infections that it will infect our native fauna population, and so it represents a very serious threat for the future inAustralia.

In Australia, while we are spared the effects of some of the clinically damaging parasites, still have an enormous impact. Australia's meat and wool industries produce about $13 billion worth of products every year, and bring in $7 billion in export earnings. Part of that is largely to do with our reputation for disease-free livestock. But worm infestation in Australia accounts for about $220 million a year, and this figure is set to increase dramatically due to drug resistance. In fact, it has been estimated that in 10 years' time there might be no drugs available to treat these worms, in which case sheep farming would become economically unviable in many areas of Australia.

In terms of human infections in Australia, Toxoplasma is a parasite that infects a wide variety of Australians. About a third of you in this room will have been exposed to Toxoplasma and some of you will have Toxoplasma in your brain. For most of you that won't present a problem throughout your life, but if, for example, you developed AIDS or you were undergoing surgery which involved immunosuppressant drugs, you could die from fatal encephalitis. Toxoplasma is spread by infected cat faeces and through contaminated meat products. If a woman becomes infected while she is pregnant, the child can die or develop severe disabilities. Even if the child has no disabilities at birth, a large percentage will go on to develop mental retardation or hearing defects, and 90 per cent will develop eye problems. And, as I said, if the immune system is compromised it can be fatal.

The estimated cost in Australia is about $1 billion per year.

Hookworm
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Hookworm is another important parasite in Australia. This [in picture on slide] is the oral part of the parasite, and it causes a lot of damage when it actually latches onto the host, due to the blood loss at the site. It is endemic in our indigenous Australian communities.

'Bali Belly'
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If you have ever had 'Bali Belly', this [in slide] is the parasite that causes it – a beautiful parasite, with these caudal flagella for movement and this ventral disc that it uses to stick on to your intestine and actually coat it.

The key to dealing with parasites is actually understanding them, and often this involves fairly basic research: how they are transmitted, what their life cycle is, which drugs will work and which parasites are the major threats to Australian livestock and wildlife.

Cryptosporidium
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I am going to talk specifically about Cryptosporidium, which is the parasite I work on. It is a fantastic parasite: it is resistant to most known disinfectants, it is the most common non-viral cause of diarrhoea worldwide. When you ingest it, sporozoites come out of the oocyst and invade the epithelial cells of the intestine. There are no really effective drugs to treat this parasite..

Cryptosporidium
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Cryptosporidium is a big problem with people whose immune systems are compromised. People can literally die of diarrhoea: they can lose over 13 litres of fluid a day and people just can't be rehydrated fast enough.

Cryptosporidium and water
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Because of the small size of the parasite – it is only about 5 microns in size – and because it is resistant to most known disinfectants, including the levels of chlorine in drinking-water, it is a very big problem in the water industry and has been responsible for numerous waterborne outbreaks. The biggest outbreak occurred in Milwaukee in 1993, where 400,000 people became infected and 60 people died as a result of drinking infected water. In fact, Milwaukee very much changed the water industry, and it is now a criminal offence in the UK to supply drinking-water that has more than one Cryptosporidium oocyst per 10 litres. So the actual detection of Cryptosporidium has become a very big industry worldwide.

When I first started working on Cryptosporidium in 1992 we really knew very little about it. We thought there was maybe one species that infected everything. We now know there are over 14 species and another 30 genotypes have been described, which are likely to be described as individual species in the near future. One of the problems with diagnosis is that most Cryptosporiidum sp.  look the same, but only two of these, C. parvum and C. hominis, are actually infectious to humans and if you find it in the water, deciding whether or not you call a boil-water advisory is very important, because more people get hurt from scaldings when you call a boil-water advisory than actually become infected with enteric parasites. So it is very important to know what you are dealing with.

Cryptosporidium diagnostic
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During my PhD I developed a diagnostic which could directly detect the two main species that cause disease in humans, using a PCR tests to amplify up the DNA which is very specific and sensitive. But what we are trying to develop is a third-generation diagnostic test that is often referred to as Lab-on-a chip.

Lab-on-a-chip
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Basically, it is a silicon chip that does all the things for detecting the parasite that we would normally do in the lab because you have miniaturised it, you can automate it, and also because you are using minute quantities of reagents there are greater reagent savings.

Lab-on-a-chip
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A Lab-on-a-chip consists of a network of microchannels etched onto the surface of the glass, and electrodes are placed at strategic locations along the chip. The sample is injected; it will be propeled along a particular route using electric currents, past a reservoir that will squirt out various reactants that will, for example, lyse the cell, past another set of reactants that will, for example, add the reagents to do the PCR and so on.

Lab-on-a-chip and PCR
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It is now possible to do PCR on a chip. PCR involves cycling DNA samples, along with specific enzymes and other components, through defined temperatures. You can propel the samples through defined temperature zones on the Lab-on-a-chip, so you can actually do the PCR on the chip as well by passing the amplified products over probes which will detect it and give off a fluorescent signal. So it is possible to amplify and detect the parasite on a Lab-on-a-chip.

One of the other advantages is that you have got up to eight input channels, so you can actually have a variety of tests going on. You can have a generic test to see if it is Cryptosporidium, you can have another test to see if it detects C. hominis or C. parvum, another test that will actually sub-genotype, because we know there are different sub-genotypes that only affect humans or only affect animals that you can sub-genotype, and then you can combine it with detection of other important waterborne parasites such as Giardia..

Cryptosporidium life cycle
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Another research area: this research is being conducted by Nawal Hijjawi, who was a PhD student and is now a postdoc at Murdoch University. She was studying the Cryptosporidium life cycle, and identified these very interesting novel stages and we wanted to see if it was actually Cryptosporidium. We wanted to be able to pick up an individual cell and flip it into a tube.

Laser tweezers (Click on images for larger versions)

We used laser tweezers to actually isolate an individual cell. Basically, laser tweezers can pick up an individual cell and flip it into a tube. So she used the laser tweezers to pick out a particular cell and then we amplified it by PCRand sequenced it, and showed that it was in fact Cryptosporidium. These lifecycle stages are similar to gregarines which are very primitive parasites that infect invertebrates.

So who cares? What difference does it make? It actually makes a very big difference, because if it is a gregarine or closely related to gregarines, it raises the question of whether Cryptosporidium can infect aquatic invertebrates and multiply in the water, or whether it can actually survive and multiply in the water. Some preliminary experiments that we have done show that it can, and so this raises enormous questions for the water industry, and also in terms of current diagnostics, which is antibody based in terms of cross-reactivity with other gregarines.

As I mentioned before, for chemotherapy there have been over 500 drugs tested against Cryptosporidium. While some have alleviated symptoms, it is actually very difficult to get rid of the parasite completely. What will often happen is that once the individual becomes stressed again, the disease will keep reoccurring.

Again, drugs against the trypanosomes are problematic. They are often toxic, and new drugs are urgently required.

In collaboration with a company called Phylogica we have been involved in developing peptides as drugs. To actually develop a drug, to get it to market level, is enormously expensive – to get it through all the FDA approvals et cetera – and in the post-genomic era, now that the Cryptosporidium genome has been sequenced, we can apply a more targeted approach and try and look at disease-specific protein targets.

Phylomers
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Peptides have had a bit of bad rap in the past because they have been developed as synthetic peptide libraries, which really did not represent the sorts of structures that you see in nature. What Phylogica have done is to develop what they call phylomers, which are short peptides that they have generated from representatives of the major categories of these organisms, and so they are much more likely to represent natural structure domains that you would find in the environment.

Phylomers
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So they are far superior. You screen a lot less because they represent natural structures, and they present an opportunity for us to actually develop specific drugs against these parasites.

One of the targets we have chosen is tubulin. It is a good target for all protozoans; it comprises as much as 10 per cent of the protein content of many protozoa. It is highly conserved, and very importantly it is very different from mammalian tubulin, thus reducing the chances of human toxicity. So that makes it a good potential as a drug target.

Screening for blockers of protein interactions
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So what we have been doing is using a Yeast Reverse two Hybrid System,. Itworks best when you are looking at two different types of proteins. So we are looking at a polymerisation of αalpha and beta tubulin.

With this system, when the alapha and beta tubulin interact, it switches on a death gene and the yeast cell dies. When a peptide interrupts the binding, the death gene doesn't get switched on and the yeast cell survives. You can use this as a screening mechanism to identify peptides which can disrupt the vital tubulin interaction in proteins, and we are working with Phylogica to use this as proof of principle on Cryptosporidium and the trypanosomes.

If this works, then we would like to use it to develop therapeutics for a wider range of parasites. So there is a potential for broad-spectrum therapeutic agents that are effective against a wide range of parasites.

Ironically, at a period when some of the threats to Australia are the greatest – for example, it has been predicted that, with climate changes, levels of parasitism amongst our closest neighbours are going to increase and so the threats to Australia from 'exotic' parasites are probably at their greatest at the moment – and the increasing drug resistance is a major problem that we face in this country, which is going to have massive ramifications for both humans and animals, we are facing a period in Australia where parasitology research is on the decline. Many facilities around the country have been downsized, and there are fewer and fewer parasitologists being trained. There are virtually no medical parasitologists being trained. It raises serious issues for our ability to control parasites in the future. While we are developing more sophisticated methods to control parasites, the issue of whether we are actually going to continue to train parasitologists in Australia needs to be addressed in future.

I would like to thank the Australian Academy of Science, all my colleagues at Murdoch University, Sydney Water – who I work with a lot, Phylogica, and the Centers for Disease Control, in the USA, who are my major collaborators internationally.

Some of the information presented in this talk, particularly about the other parasites, is available on the Parasitology Network website at www.parasite.org.au/arcnet.index.htm.

Questions/discussion

Question: Apart from drug resistance, how rapid are mutations in the different species of parasites? And are new subtypes developing relatively rapidly that potentially would be able to infect humans but currently do not?

In terms of a parasite changing from a non-zoonotic to a zoonotic, I don't see that happening very readily. But certainly the parasites are developing rapid resistance. You can generate resistance very quickly and easily in the lab, and in the field situation we are seeing that happening all the time.

Question: This is a naive question, but I can't imagine any drug companies spending vast sums of money on diseases which affect mainly people in underdeveloped countries. This is clearly public-good research. Where is your source of funding, and where do you expect to find widespread support for this very important area?

This is one of the problems with parasitology research: it is not terribly sexy. A lot of the funding actually comes from water authorities, because it is in their best interests to detect it and control it. And some international agencies are prepared to fund research into the development of drugs.

Against Cryptosporidium there wouldn't be an enormous number of drug companies out there looking to put money into developing drugs, but for some of these parasites – for example, here in Australia for Toxoplasma, for some of the worm parasites – there is an enormous market because the potential economic losses are huge. So it depends very much on the parasite.

Question: Are there also threats to Australia from endogenous parasites? Do our native animals act as reservoirs for parasites that could, for instance, cross into domestic species?

Well, for example, we know that native animals are reservoirs for Toxoplasma, and there has really been very little work done on what effect it has on the native fauna and how much of a reservoir it can be for human infection. So that is an area that really needs to be looked at. It is something that could potentially be a big threat in the future.

Question: How long are these phylomers? How long sequences are there, and do you think really they adopt a certain structure conformation, or is it just the combination of specific amino acids which is more natural, that that would account for its better effectivity that you mentioned?

They are about 150-300 bases in length, so they are fairly short. In terms of the structures that they actually develop, we don't really know but we do know that it has proven very useful. Say, for example, this company have used it on cancer research, so they have actually used it successfully to disrupt protein-protein interactions.

In terms of parasites, the main thing is just for it to be able to disrupt it. How it is actually disrupting it, we are not entirely sure.

Question: I am very interested in bottled water. It is a huge market out there. Does it offer any advantages over tap water in terms of the parasite load?

No. Basically, what is in your bottled water is just as contaminated as what is in your tap water.

Question: Years ago I heard about a terrible disease called bilharzia. It was treated by putting copper sulphate in the water, which broke up the life cycle. Where does it sit at the moment in the world?

Question (cont'd): It was a worm that developed in the gut of a person and just gradually made their energy levels drop. They eventually died.

Question: As a comment: the more commonly used word today is 'schistosomiasis'. There are three main species that are very infectious to humans. The disease is under reasonable control. China has got it under pretty good control; it is still a very major problem in Egypt and a not unreasonable problem in the Philippines. There is one wonderful drug that came out of the WHO Tropical Diseases Program, called Praziquantel. Now the big question is how soon resistance will be developed if it is widely used. There is one vaccine in clinical trial, from France, but we don't yet know how well it will work.

Question: Peptide drugs might be useful, I could see, in something that stayed in the gut. But if it is in the bloodstream, some peptides get broken down before they get into the bloodstream. What is the delivery system, or how do you engineer a peptide so that it actually gets into the bloodstream if you are targeting a parasite in the bloodstream?

Well, with Cryptosporidium it is relatively easy. We can just get protein transduction domains across the membrane quite easily. With the trypanosomes the major focus was really to understand protein-protein interactions and we will be injecting the peptides into the bloodstream.

Question: I think you spoke of the standard adopted in Milwaukee for drinking-water of one particle per 10 litres.

This is legislation that has been introduced  in the UK: one Cryptosporidium oocyst per 10 litres.

Question (cont'd): I presume you have some means of concentrating these for determination and analysis. How do you separate them out?

Basically, 20-litre samples are taken and they are put through a filter, and then what is on the filter is eluted off and coupled with magnetic beads that have an anti- Cryptosporidium antibody to them. They are purified further and then they are taken off the magnetic beads and detected with a fluorescent antibody. But it is highly problematic. The recovery rates are hugely variable, and the UK industry is very upset about this regulation being put in place when they don't really have very good methods of actually detecting it. It is a very big issue in the industry at the moment.

Question (cont'd): It was the matter of the filtration that puzzled me. How reliable is that?

As I said, there are a couple of different approved filtration methods, but it is extremely variable. Cryptosporidium is only about 5 microns in size, and often, if the pressure gets too high, they can actually burst on the membrane. And they tend to be very sticky so it is very difficult to get them back off the filtration membrane. So it is very problematic. The recovery rates can vary from 5 per cent to 60 per cent.