The venom of the platypus (Ornithorhynchus anatinus)

The platypus (Ornithorhynchus anatinus) and its venomous characteristics in particular, have experienced a long history of being treated with disbelief or ignored completely. For example, the first platypus specimen sent to Britain in 1798 was thought to be a fake, stitched together by a taxidermist using mammalian parts and a duck bill (Grant, 1995). Even a recent review entitled "Venomous Mammals" (Dufton, 1992) makes no mention of the platypus and a recent case report of platypus envenomation (Tonkin and Negrine, 1994) in the British Journal of Hand Surgery was accompanied by the following, apparently facetious, Editor's note: "A spate of Dodo bites has recently been recorded in a remote area of New South Wales and will be reported by Dr Tonkin in the next issue of the Journal". This is despite the fact that several publications exist which deal specifically with the subject, most notably, Martin and Tidswell (1895), Kellaway and LeMessurier (1935), Calaby (1968), Temple-Smith (1973) and Fenner et al. (1992).

The venom apparatus of the platypus is restricted to the male and externally consists of a pair of movable calcaneus spurs on each hind-limb. Each spur normally lies against the limb but is attached at its base to an articulating bone which allows it to be erected at right angles to the limb when required (Temple-Smith, 1973). At the base of the spur is a sac or reservoir from which a tiny duct extends through the spur. A large, distensible duct, which superficially traverses the biceps muscle, connects the reservoir to the crural venom gland. There appears to be a temporal relationship between breeding status and crural gland secretory activity.

Although the clinical manifestations of platypus envenomation have been systematically investigated in only two cases (Fenner et al., 1992; Tonkin and Negrine, 1994), many anecdotal accounts exist and provide a source of consistent information on post-envenomation sequelae. The earliest documented case of envenomation appears to be that of Jamieson (1817, cited in Martin and Tidswell, 1895), who described the effects of envenomation: "[the platypus] stuck its spurs into the palm and back of his right hand with such force, and retained them in with such strength that they could not be withdrawn until it was killed. The hand instantly swelled to a prodigious bulkThe pain from the first was insupportable[he] did not recover the perfect use of his hand for nine weeks". The extensive swelling and lasting pain described by Jamieson are consistently reported in most of the subsequent anecdotal accounts. For example, Spicer (1876) cites a case where "The pain was intense and almost paralysing. But for the administration of small doses of brandy, he would have fainted on the spot: as it was, it was half and hour before he could stand without support: by that time the arm was swollen to the shoulder, and quite useless, and the pain in the hand very severe". Martin and Tidswell (1894) report a similar case: "Mr. E. all the time suffered intense pain, and presently the wounded finger, then the hand, and ultimately the whole arm up to the shoulder swelled to a serious extent". Temple-Smith (1973) confirmed these anecdotal reports of the painful effects of platypus envenomation by injecting a small quantity into his forearm. This resulted in "immediate intense pain which diminished to a dull muscular pain, impairing use of the arm for 48 hours."

Fenner et al. (1992) provided the first clinical case report of platypus envenomation. They report that the affected patient, who received spur wounds to the hand, presented with oedema and lasting severe pain which did not respond effectively to morphine and was only alleviated following a wrist block. Laboratory blood tests revealed an increased erythrocyte sedimentation rate (ESR), indicating possible coagualopathy. The pain in this case persisted for several months and substantially impaired use of the affected limb. Intense pain and oedema were similarly described in a more recent case reported by Tonkin and Negrine (1994), although the symptoms in this case subsided within several weeks.

From the anecdotal accounts and the clinical case studies, it is apparent that pain and oedema are the major symptoms associated with platypus envenomation. There does not appear to be any evidence of systemic neurotoxicity, myotoxicity (although it should be noted that Fenner et al. (1992) reported "significant" forearm muscle wasting in the affected patient two weeks after envenomation but it was not established whether this was due to disuse of the affected limb because of the severe pain or caused directly by the venom) or necrotic effects and there have been no reported human fatalities.

The earliest experimental studies on the venom were conducted by Sir Charles Martin and Dr Frank Tidswell towards the end of the 19th century (Martin and Tidswell, 1895). They expressed secretion from excised crural glands and found that it produced localised swelling and tenderness when injected subcutaneously into a rabbit. When three rabbits were injected intravenously, a rapid fall in blood pressure, respiratory distress, including "expiratory convulsions", and death followed. Post-mortem examination revealed that two animals probably died from extensive intravascular coagulation. However, blood from the remaining animal, in which the material was injected more slowly (and which died somewhat later than the others), exhibited no signs of clotting in vessels. In fact a sample of this animal's blood was found to clot abnormally slowly. Martin and Tidswell concluded that the effects of the venom on blood pressure, coagulation and tissue oedema were analogous to those produced by Australian Snake venoms.

Kellaway and LeMessurier (1935) extended the initial observations of Martin and Tidswell using material supplied by Martin which was "probably 30 years old". This was injected intravenously into two rabbits and resulted in severe dyspnoea. Using freshly obtained material, Kellaway and LeMessurier demonstrated it to be feebly haemolytic but found that it produced a rapid and profound fall in blood pressure and death when intravenously injected into rabbits. The fall in blood pressure was associated with peripheral vasodilatation. In one injected rabbit, particles of "whipped-out fibrin" were found in the heart upon post-mortem examination, consistent with the pro-coagulant activity observed by Martin and Tidswell. Kellaway and LeMessurier further tested the material using several in vitro preparations. They found that the venom produced vasodilatation in the perfused rabbit ear and in cat mesentery, supporting the notion that the fall in blood pressure was peripheral in origin. The venom also contracted the isolated guinea pig uterus and rabbit jejunum.

Temple-Smith (!973) investigated the composition and physiological effects of the venom as well as the seasonal dependence of its secretion. He found that when the venom was injected subcutaneously into mice, some squealed and were observed to lick, scratch and bite the injection site. These behavioural symptoms are typical indicators of pain and are sometimes used quantitatively in behavioural studies of nociception. In Temple-Smith's investigation, the venom was fractionated and fractions assayed for both lethality (following i.v. injection) in mice and "cutaneous activity" (plasma extravasation). Lethality was associated with high-molecular weight material. Temple-Smith found that mice which had received lethal or sub-lethal doses of venom typically exhibited hyperventilation, convulsions, cyanosis and apparent hind-limb paralysis. Post-mortem examination revealed no tissue or vascular damage except in the lungs where vascular damage, constricted alveoli and oedematous septae were evident. In addition, large accumulations of erythrocytes were observed in alveolar capillaries, suggesting blood flow through the lungs had been reduced by obstruction of the pulmonary arteries. Temple-Smith also demonstrated that the venom contains hyaluronidase and proteolytic activities.

The smooth muscle activity has been investigated further (de Plater et al., 1998a) and it has been demonstrated that the venom produces a relaxation of the rat uterus in vitro. This appears to be the effect of a natriuretic peptide in the venom. Natriuretic peptides are a family of peptides which are highly conserved between species. In mammals, three classes have been described: atrial natriuretic peptide (ANP) which is predominantly produced by the cardiac atria; brain natriuretic peptide (BNP), originally isolated from the brain but predominantly produced by the cardiac ventricles; and C-type natriuretic peptide (CNP), the distribution of which includes the brain and endothelium. It is the latter (CNP) which is found in platypus venom. Although the normal physiological role of natriuretic peptides, in particular CNP, is unclear, their natriuretic, diuretic and vasorelaxant properties suggest that they play a role in the control of blood pressure and fluid/salt homeostasis. The natriuretic peptides, including the platypus form, elevate cyclic GMP in vascular smooth muscle, resulting in vasodilatation. It is thus possible that the platypus natriuretic peptide is responsible for the hypotensive effect of the venom in experimental animals described above. The platypus (and other) natriuretic peptides release histamine from mast cells and this may produce further vasodilation and contribute to some of the local effects of envenomation, such as oedema.

The finding (Murayama et al., 1997) that CNP is present in the venom of the South American pit viper (Bothrops jararaca) is the only other example of a CNP in an animal venom, although the Green Mamba snake (Dendroaspis angusticeps) venom contains an ANP-like peptide.

Although the endogenous function of CNP is unknown, the fact that CNP is released from endothelial cells following stimulation with cytokines suggests it may serve as an autocrine or paracrine mediator in cytokine-associated disorders (Suga et al., 1993). Also, patients with septic shock and chronic renal failure show elevated serum CNP levels (Hama et al., 1994). These findings, in addition its aforementioned effects and discovery in platypus and pit viper venom, implies a pathophysiological function.

Local pro-inflammatory effects such as the release of amines from mast cells (e.g. by CNP), are unlikely to be responsible for the severe and lasting pain experienced by platypus-envenomed patients because these effects usually produce relatively mild and short-lived symptoms. The effects of a bee sting, for example, which predominantly result from mast cell amines, resolves relatively quickly (in the absence of significant anaphylaxis).

The possibility that platypus venom may have a direct effect on nociceptive (pain-sensing) sensory neurones, rather than acting via inflammatory cells, is currently being investigated (de Plater et al., 1998b). Unfortunately, it is impossible to gain access to and electrical control over nociceptive terminals in the periphery to address this question directly. Instead, the cell bodies of nociceptive neurones which are found in sensory ganglia may be used as a model of the nociceptor terminal. These cell bodies express properties associated with their nociceptive terminals. For example, they are activated by pain-producing inflammatory mediators and capsaicin (the pungent component in chillies which gives rise to their burning effects).

A short (10 second) application of platypus venom to these neurones at pH 7.4 has little or no effect. At pH 6.1, however, the venom produces large inward currents which last for several minutes. pH 6.1 alone produces a single inward current (believed to underlie the painful effects of acidic pH) but this is transient and lasts, at the most, for as long as the low pH is applied (e.g. 10 seconds for a 10 second application). This low-pH current is markedly potentiated by inflammatory mediators (Kress et al., 1997), which is particularly interesting given that inflammatory exudates are acidic (McCarty et al., 1966). It is conceivable that the pro-inflammatory effects of platypus venom may produce local changes in pH sufficient to both potentiate the low-pH current and activate the lasting inward currents described above. Inward currents, which depolarise neurones, make them fire action potentials directly or make them more likely to fire action potentials. Occurring at peripheral nociceptor terminals, this would ultimately give rise to a sensation of pain or increased sensitivity to painful stimuli. The component or components of the venom which produce this effect are not known and further work is under way both to determine this and to elucidate their mechanism of action.

An understanding of how platypus venom produces its severe, lasting painful effects (which may be refractory to morphine - see above) may facilitate our understanding of mechanisms underlying analogous chronic pain states (e.g. some causalgias and cancer-associated pain syndromes) for which no effective analgesics are available. This could potentially lead to the discovery of novel pharmacological targets.

For more information on the platypus, contact Greg de Plater at the Australian National University

References

Calaby, J. H. (1968) In Bücherl, E., Buckley, E. E. and Deulofeu, V. (Eds.) Venomous Animals and their Venoms. Vol 1: 15-29.

de Plater, G. M., Martin, R. L. and Milburn, P. J. (1998a) Toxicon. 36: 847-57.

de Plater, G. M., Martin, R. L. and Milburn, P. J. (1998b) J. Physiol., 506.P: 154P.

Dufton, M. J. (1992) Pharmac. Ther. 53: 199-215

Grant, T. (1995) The platypus: a unique mammal. UNSW Press. Sydney

Hama, N., Itoh, H., Shirakami, G., Suga, S., Komatsu, Y., Yoshimasa, T., Tanaka, I., Mori, K. and Nakao, K. (1994) Biochem. Biophys. Res. Comm. 198: 1177-82.

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Murayama, N, Hayashi, M. A. F., Ohi, H., Ferreira, L. A. F., Hermann, V. V., Saito, H., Fujita, Y., Higuchi, S., Fernandes, B. L., Yamane, T. and de Camargo, A. C. M. (1997) Proc. Natl. Acad. Sci. USA. 94: 1189-93.

Spicer, W. W. (1876) Pap. Proc. R. Soc., Tas. 1876: 162-67.

Suga, S. -I., Itoh, H., Komatsu, Y., Ogawa, Y., Hama, N., Yoshimasa, T. and Nakao, K. (1993) Endocrinology. 133: 3038-3041.

Temple-Smith, P. D. (1973) Seasonal breeding biology of the platypus, Ornithorhynchus anatinus with special reference to the male. PhD Thesis, Australian National University, Canberra. Australia

Tonkin, M. A. and Negrine, J. (1994) J. Hand Surg. 19B: 162-64