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TOXICOLOGY and TOXINOLOGY of Ixodes holocyclus

What are the toxins?
What are the toxic pathways?
Why does a paralysis toxin(s) exist?
Current research on toxins and toxoid vaccines
Summary of Cooper's findings
Some thoughts about toxic mechanisms
Bibliography

What are the toxins?

It is believed that the physical effects seen in tick paralysis are caused by various neurotoxins produced in the salivary glands of engorging ticks.

Originally, Stone et al (1983) isolated several toxic fractions from the salivary glands of Ixodes holocyclus, including a protein neurotoxin (originally named holocyclotoxin) that causes paralysis, and another toxin that is lethal but non-paralysing (Malik and Farrow, 1991). The "holocyclotoxin" was described as a protein and the most toxic fraction had a molecular weight of 40,000-60,000 D. It was a relatively stable compound, especially when freeze-dried. The holocyclotoxin affected efferent pathways (neuromuscular transmission) whilst the lethal toxin may have affect other neural pathways- for example afferent pathways and the autonomic nervous system. Stone also recovered toxin from an artificial medium into which Ixodes holocyclus females salivated following attachment to a silicone rubber membrane. Much of the work done on the feeding behaviour of the tick was done by Stone who used electrodes implanted into the ticks to study the cyclical patterns of feeding, salivating and resting (Fitzgerald, 1998).

More recently, researchers at University of Technology Sydney (UTS) under Associate Professor Kevin Broady of the Immunobiology Unit have isolated 3 low molecular weight toxins and demonstrated that all 3 can cause hindlimb paralysis in baby mice. This was achieved by a simple and ingenious experiment by a PhD student using radiolabelled tick extracts and pinched off nerve endings. This information changed the purification methods and with some further innovative work the toxins were purified. Only 1 could be isolated in quantities which have permitted further research. The molecular weight was relatively small at 6000 D, with approximately 50 amino acids, homologous to scorpion and spider toxins. Partial amino acid sequence data has been used to design PCR primers which have been employed to isolate the gene for one of the toxins. Research is currently underway to sequence the genes for the other tick neurotoxins. Synthetic neurotoxins will be produced by recombinant DNA and solid phase peptide synthesis techniques and investigated for vaccine potential. Professor Broady suspects that the 3 toxins are closely related and may just represent post-translational modifications of the same molecule. He suspects that it is possible that one toxin may cause all the different effects associated with the paralysis syndrome. "Hopefully we will be able to test this when we have large amounts of active recombinant toxin. If there is a second type of toxin, it will mean going back to the start of our work and eliminating the effect of the toxin we are now working on" (K. Broady, pers com).

It has been suggested that the toxin causing cardiac dysfunction is not inactivated by hyperimmune serum, suggesting that it is not an immunogenic toxin (this information unofficially presented at a tick information seminar presented by Dr R Atwell, Wollongong 2001).

A lesser known toxin, which is deep red, water-soluble and produced by the cuticle of the fully engorged larvae, nymph and adult female has unknown significance. It appears from the time the tick drops off the host until either ecdysis or oviposition occurs and is secreted through the cuticular canals. The substance can be collected by simply washing engorged females in water. When injected intraperitoneally into mice it causes respiratory paralysis within 9-10 hours (Jones, 1991).

Anticoagulants. See: Anticoagulant in the tick Ixodes holocyclus.

What are the toxic pathways?

Acetylcholine release at the motor end plate is reduced in tick paralysis (Cooper, 1976), possibly as a result of diminished calcium entry into motor nerve terminals, or some other interference with presynaptic excitation-secretion coupling. The effects on neuromuscular transmission have been shown to be temperature dependent in vitro being more pronounced at higher temperatures. This corresponds with clinical and experimental observations that high ambient temperatures adversely affect the clinical course of the disease.

Axonal conduction is unaffected in paralysis caused by Ixodes holocyclus. Interestingly, in Cooper's experiments where nerve-muscle preparations were incubated in solution containing toxin the paralysing effect was delayed six to seven hours after the addition of toxin. For a discussion on the toxic mechanisms, see the extract from FitzGerald (1988) below.

Why does a paralysis toxin exist?

There has been some discussion of this in the literature. Does the paralysing toxin serve a purpose in aiding the survival of the paralysis tick or is it vestigial, an evolutionary remnant? Some of the theories:

• Paralysis as a side effect of another effect still beneficial to the tick? Given that the paralysing effects on native hosts are generally neglible in the natural setting the toxin's paralysing effects seem unnecessary. However, this still leaves the possibility of other useful but non-paralysing effects of these molecules. They may, for example, have a role in the feeding process, or in local anaesthesia in the host.
  
• Paralysis as an effect currently beneficial to a parasitic existence. For example a paralysed host may be less likely to wander out of the desired niche environment. The tick also be less likely to be brushed off in a paralysed animal
• Paralysis toxin as a remnant effect once beneficial to a parasitic existence? Perhaps there was once a role with native animals whose evolutionary development has outstripped that of the tick to overcome the problem by developing immunity. On the other hand, because nearly all native mammals seem equally well adapted perhaps they have a generally improved immune system not directly selected by pressures from the paralysis tick.
• Paralysis toxin as a remnant once beneficial to a predatory existence? Here I quote Assoc Prof Kevin Broady- "Ticks being arachnids are related to spiders and scorpions and mites. Spiders and scorpions have retained toxins and developed specialised delivery structures [fangs and telsons] while mites and ticks have lost this feature. Of the 800 species of ticks, only 40 have been reported as causing a form of toxosis . So as ticks have moved from a predatory existence to a parasite existence most species have lost their toxin which would have been a disadvantage for a parasitic lifestyle. The Australian tick of course would be the most backward and still retain an effective toxin [ but it still manages to survive]. Our gene sequence of the tick toxin shows high homology to scorpion toxins so this hypothesis has some credence."
  
• Combination of reasons above. For example, the toxin may have once had a predatory role but now have a parasitic role. Or it may have had a predatory role, then a parasitic role and now no role, in which case it should eventually be doomed to the evolutionary scrap heap.

Current Research

Research is being undertaken by a team led by Associate Professor Kevin Broady at University of Technology Sydney (UTS) concerning tick toxins and the making of an anti-tick toxin vaccine- go to Departments section, then to Cell and Molecular Biology and then Research. See also "Vaccine against the neurotoxins of the Australian paralysis tick, Ixodes holocyclus." Associate Professor K.W.Broady Ph: 61-2-9514 4101 Head, Department of Cell and Molecular Biology Fax: 61-2-9514 4026 University of Technology, Sydney, Email: kevin.broady@uts.edu.au . See also the page Current Research.

Summary of Cooper's experimental findings

The following is an extract from Fitgerald (1998) summarising the experimental work of Cooper (1976):

"Cooper's (1976) work on the pathogenesis of Ixodes holocyclus poisoning was the first attempt to discover the mechanism by which the toxin/s caused paralysis. Cooper's neurophysiology studies in dogs showed a marked reduction of muscle compound action potentials evoked by nerve stimulation compared to that of control dogs. No change could be demonstrated in the action potential or maximum conduction velocity of the peripheral nerves. He concluded that there was a disturbance of neuromuscular transmission occurring at or near the neuromuscular junction.

A chance observation that affected mice kept in high ambient temperatures had reduced survival times led to the realisation that there might be an effect of temperature on the development of the paralysis. He then went on to show that nerve-muscle preparations from paralysed mice exhibited a temperature dependent paralysis in vitro- because at temperatures at which muscles failed to respond to nerve stimulation there was a normal response to direct muscle stimulation. This effect was demonstrated to be reversible. This could prove to be an important clue in resolving the exact mechanism of action of the toxin because any physiological mechanism on which the toxin exerts its effect should show a similar response to temperature changes.

Cooper then showed that a reduction of end-plate potential amplitude seen in tick paralysis is due to a decrease in the amount of acetylcholine released in response to a nerve action potential. As quantal size was not reduced, the reduction in evoked neurotransmitter release in tick paralysis must be due to a temperature-dependent decrease in quantal content of the end-plate potential. Because spontaneous release still occurs, it seems unlikely that the toxin actually blocks the neurotransmitter release site. Cooper therefore concluded that the toxin somehow interferes with the depolarisation-secretion coupling mechanism- ie the nerve's action potential does not initiate secretion of neurtransmitter. One possibility is that it blocks the influx of Ca2+ ions which follow depolarisation.

Coopers post-mortem studies on dogs which had died of tick poisoning, showed both gross and histologic evidence of some pulmonary congestion and oedema. This was more pronounced on the side on which the dog was lying during the terminal stages of the disease. In those dogs in which the condition was allowed to progress to death (some had been euthanased in the terminal stages, several small haemorrhages were found scattered throughout the brain. These did not show any constant relationship to any particular areas. He concluded that these were secondary to hypoxaemia in the terminal stages. Pulmonary congestion was thought to he a secondary change.

In experiments designed to demonstrate whether the crude toxin was active in vitro using nerve-muscle preparations from mice. Cooper showed an identical temperature dependent reduction in response in both phrenic nerve-diaphragm and soleus muscle preparations to that seen in adult mice with adult female I. holocyclus.

It is very interesting to note that in these experiments where nerve-muscle preparations were incubated in solution containing toxin there was a delayed effect for six to seven hours after the addition of toxin. This effect was again temperature dependent. Mice injected with toxin do not develop signs of paralysis for some 8 to 12 hours after the injection. Possible explanations for this include: (a) that the toxin has to be modified by the host in some way by the host animal before becoming effective or. (b) that the delay in onset of action of the toxin is due to simple delay in reaching its site of action. (Given that the 6000 dalton MW molecule must leave the intravascular lymphatic compartment and travel to the neuromuscular junction crossing presumably several different membranes.) It is interesting to relate this experimental observation to the well recognised clinical phenomenon of slowness in onset of action and also slow-ness in resolution of effects of the toxin. Another interesting feature of clinical tick poisoning is the delay (commonly felt by practitioners to be approximately 12 hours) before signs of improvement are seen after administration of hyperimmune antitick serum. This can be compared to the much more rapid rates of recovery from presynaptic paralysis seen for example in some elapid snake envenomations where hyperimmune serums are administered. Clearly, there remains much to be resolved about the toxicodynamics of holocyclotoxin i.e. how it travels to target sites, how, where and for how long it is bound and also whether bound toxin is able to be accessed and neutralised by the globulins in antitick serum. It would also be helpful to know more about the pharmacodynamics of the globulins in the tick serum as the delay in response in clinical cases often results in simple cases becoming complicated and complicated cases becoming fatal.

Cooper concluded from this experimental data, that the abnormality of transmitter release could describe all of the reported signs of tick paralysis with the possible exception of vomiting. Although no specific treatments emerged from his studies the finding of the temperature dependence of the toxin was important in that "controlled hypothermia" is now generally accepted as beneficial to recovery. Virtually nothing is known at present of the effects, if any, of the toxin on acetylocholine release from paraysmpathetic postganglionic autonomic neuroeffector junctions. If autonomic dysfunction is involved in tick poisoning, this could contribute to some of the variability and complexity seen in practice. Also, the possibility raised by Cooper's work that nerve terminals innerating red muscle fibres are more susceptible to tick paralysis than those innervating white muscle fibres needs further investigation."

Some thoughts and questions about toxic mechanisms [NF]

Bibliography

Anastopoulos P; Thurn MJ; Broady KW; Department of Cellular Pathology, University of Technology, Sydney, Broadway, New South Wales.Anticoagulant in the tick Ixodes holocyclus. Aust Vet J, 1991 Nov, 68:11, 366-7

Jones DK: Tick Paralysis; in JD Stewart Memorial Course for Veterinarians: Proceedings 149: Emergency Medicine and Critical care, The Post Graduate Committee in Veterinary Science, University of Sydney, 1991.

Malik R, Farrow, BRH: Tick Paralysis in North America and Australia, in The Veterinary Clinics of North America, Small Animal Practice, Vol 21:1 Tick Transmitted Diseases, 1991.

Masina, S. (1995) BSc(Hons) Thesis, UTS

Masina, S., Thurn M.J. and Broady, K.W. (1997) Gene sequence of an Australian tick neurotoxin. Abst 12th World Congress on Animal, Plant and Microbiol Toxins, Cuernavaca, Mexico, September 1997.

Stone BF, Binnington KC, Gauci M and Aylward JH (1989) Tick/host interactions for Ixodes holocyclus: role, effects, biosynthesis and nature of it's toxic and allergic oral secretions. Experimental and Applied Acarology 7 58-69.

Thurn,M.J. and Broady , K.W. Characterisation of the Toxin from Ixodes holocyclus .(Abst.) Toxicon , 28(3), 257 (1990)

Thurn,M.J. and Broady, K.W. A Tick Toxin. In: Toxins and Targets. (1992) D.Watters, M.Lavin, D.Maguire and J.Pearn (eds). Harwood Academic Publishers, N.Y., p75.

Thurn,M.J., Gooley,A. and Broady, K.W. Identification of the neurotoxin from the Australian paralysis tick, Ixodes holocyclus. In: Recent Advances in Toxinology Research, Vol.2, (1992)P.Gopalakrishnakone and C.K.Tan (eds). National University of Singapore, p243.

Thurn,M.J. (1989 ) BSc(Hons) Thesis, UTS

Thurn,M.J. (1995) PhD Thesis, UTS

 

 

 

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