Pharmacological and immunological studies aimed at prevention of Pimelea poisoning of cattle

Aims to determine whether the toxins of Pimelea can be immunogenic and if so, to investigate whether antibodies raised to the toxins offered any protection against Pimelea toxicity. The study also provides opportunity to examine the in vitro contractile effects of the toxins on ovine pulmonary venule preparations.. Poisoning by Pimelea plants causes annual production losses exceeding $10 million to the Queensland cattle industry. Poisoning is caused either by ingestion of plants or by inhalation of dried plant debris. Daphnane and tigliane diterpenoid toxins found in the various Pimelea species are absorbed into the blood and cause pathological responses in target tissues through prolonged activation of protein kinase C isoenzymes. Simplexin, huratoxin and derivatives are the most toxic compounds prevalent in Pimelea species causing poisoning of cattle. Cattle are more vulnerable than sheep and horses to the toxins because the bovine pulmonary venous system has distinct sphincter -like arrangements in the smooth muscle coating of the venules. Prolonged contraction of the pulmonary system leads to oedema, jugular distension and right failure. At the present time there is no effective means of controlling pimelea poisoning in cattle, apart from removing animals from pastures infected with the plant. The main objectives of the research program described this thesis was to determine whether the toxins could be made immunogenic and if so, investigate whether antibodies raised to the toxins offered any protection against Pimelea toxicity. The study also provided the opportunity to systematically examine the in vitro contractile effects of the toxins on bovine pulmonary venule preparations. These organ bath experiments also allowed investigation of the efficacy of purified anti-toxin antibodies and potential antagonists of the toxins. The family of Pimelea diterpene toxins were isolated from dried Pimelea trichostachya plant material through a combination of solvent extraction and partition, column chromatography and preparative reverse phase HPLC. The toxins were conjugated to carrier proteins (HSA or ovalbumin) through a succinylate linker. Two structurally similar and commercially available daphnane orthoesters, mezerein and resiniferinol were also conjugated to carrier protein. Two groups of rabbits were vaccinated with the Pimelea- derived protein conjugates. ELISA methodology was developed to detect the presence of specific antibodies against Pimelea toxins from the serum of the vaccinated animals. After confirming the presence of specific IgG against Pimelea toxins in vaccinated rabbits, three groups of cattle were vaccinated with vaccines prepared from Pimelea, mezerein and resiniferinol protein conjugates. Cattle were vaccinated with the mezerein and resiniferinol conjugates to investigate the possibility of using commercially available compounds for vaccine preparation. The ELISA results for the vaccinated cattle showed the presence of specific antibodies which recognised toxin-protein conjugates coated to the plates. The ELISA cross reactivity data also showed that antibodies raised in animals vaccinated with mezerein and resiniferinol conjugates also recognised the Pimelea toxins. Vaccinated and control cattle were exposed to Pimelea trichostachya plant material to establish whether the antibodies had any protective effects. Both groups of cattle developed symptoms of Pimelea poisoning over two weeks, although these animals were dosed with much higher Pimelea concentrations than would be expected under normal grazing conditions. Various blood parameters were monitored throughout this experiment and there were some trends evident under the lower dose regimes that vaccinated animals were less affected than controls.The organ bath studies of isolated bovine pulmonary venule preparations showed that the EC50 toxin concentration required to elicit half maximal contraction (relative to the 1.0 M 5-HT response) was in the sub-micromolar range. Purified rabbit anti-toxin IgG was shown to attenuate the contractibility of bovine pulmonary venules in response to Pimelea toxins (EC5O) a dose dependent manner, whereas the purified bovine anti-toxin IgG lacked any efficacy. Nux vomica CM tincture (at doses above 200 L in the 25Ml baths) completely inhibited contraction response to toxins (EC50).