Battling the bushwhackers

Certain natural hazards attach to the otherwise pleasant business of going bush to commune with nature in the great southern land - deadly spiders, snakes and crocodiles lie in wait for the unsuspecting bushwalker.

And Australia has the world's deadliest tick, a nasty bushwhacker, Ixodes holocyclus, the bane of pet owners and veterinarians along a great arc of the eastern coast, from the Iron Range in far north Queensland, to Lakes Entrance in eastern Victoria.

The tick's natural host is the bandicoot, but it also takes a toll on cats, dogs, foals and calves. Around five days after it attaches and begins to feed on its host's blood, the tick begins secreting a potent neurotoxin in its saliva.

Some 24 hours after the venom enters its bloodstream, the animal becomes lethargic and exhibits paralysis of its rear limbs. Vomiting and unconsciousness follow, and if the tick is not detected and removed, death can occur within 48 hours.

There's a dog-serum anti-venom, but no vaccine. Developing a vaccine against any external parasite is one of immunology's major challenges.

Matt Padula has spent seven years on a part-time PhD, looking for suitable antigens for a tick vaccine since 2000. Padula is also a full-time technical specialist at the Proteomics Technology Centre of Excellence at the University of Technology, Sydney (UTS) which provides technical advice to researchers around Australia with proteomics problems.

The tick's neurotoxin seemed an ideal target, and Padula spent five years investigating its potential as a key antigen for a vaccine.

But two years ago, Dr Ben Herbert, a founder both of Sydney company Proteome Systems and the Australian Proteome Analysis Facility, joined UTS as director of the centre.

"He suggested that, rather than trying to stop something that occurs five days after the tick begins to feed, we should be hunting vaccine targets that stop the tick feeding in the first place," Padula says.

"Essentially what I've been trying to do since then is to fractionate whole male and female ticks, and see what proteins we get on our 2-D gels."

Like other ticks, I holocyclus secretes proteins in its saliva that suppress the host species' immune response - otherwise the host could begin making antibodies against the tick's proteins, with potentially lethal results.

Reasoning that protease enzymes were likely to be involved in neutralising the host's immune response, Padula and his colleagues devised a way of exploiting the high biochemical energy present in enzyme reactions to activate a fluorescence reaction, so that any spot containing an enzyme would show up in their 2-D gels.

They purified other proteins via affinity chromatography and Western blotting, using commercial anti-tick dog serum, and several litres of serum from a human subject who had developed a hyperallergic reaction to tick bites.

The anti-tick antibodies in the sera would bind their corresponding antigens, and could then be eluted and run through a mass spectrometer to determine their mass and peptide sequence.

---PB---

Tick genome

International genome and proteome databases now hold massive data sets on scores of life forms, from bacteria to mice and humans, but there is no tick genome, even though ticks are serious parasites of humans and livestock.

In the US, ticks transmit the rickettsial infections Lyme disease and Rocky Mountain spotted fever, and babesiosis in cattle. There is some evidence that I. holocyclus is the vector for a native rickettsia that causes a Lyme-like disorder in some people and pets.

Padula says the lack of a tick genome database or proteome library makes it very difficult to identify tick proteins and establish their function - Purdue University in the US has recently begun a tick genome project, but it will be some time before the database is sufficiently annotated to be useful to immunologists.

So the UTS team is doing de novo peptide sequencing of tick proteins and enzymes, trying to determine their function by matching them up in databases for other arthropods.

In addition to salivary enzymes, they are looking for other soluble proteins that may mute the host's defensive responses.

By running sequential extractions, they are extracting proteins that vary from soluble to hydrophobic. The former are likely to include allergens, while the latter may include membrane-bound proteins from tick cells.

In the 1980s, CSIRO immunologists attempted to develop a vaccine against gut antigens from cattle tick, Boophilus microplus, that would destroy the lining of the tick's gut, causing it to starve and drop off the animal.

If the UTS centre succeeds in isolating candidate tick antigens, they will probably be handed over to vaccine developers to work with. Padula is using the tick as a model for developing novel proteomics techniques that can be applied to a variety of "orphan species" for which no genome or proteome data are available - and may not be available for decades, if ever.

"The tick is an interesting organism. It forces you to think about developing interesting new ways to purify and identify proteins," he says.

The centre is running projects on a number of other economically important parasites, including the pathogenic yeast, Cryptococcus neoformans, which can cause cryptococcosis - a chronic infection in immunosuppressed individuals, with symptoms including inflammation of the heart and brain.

The centre is also comparing protein-expression profiles for normal and bleached coral polyps.

Padula's PhD is currently funded at the discretion of the centre, and he fears the project may be wound up without delivering enough candidate antigens to interest a commercial developer, when he completes his research early next year.

"A tick vaccine would save an enormous number of pets and livestock," he says. "Veterinarians are certainly interested - they're always asking me when a tick vaccine will become available."