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Development of a Cane Toad Biological Control cover page

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Development of a Cane Toad Biological Control

Robinson, T., Hyatt, A., Hardy, C. Pallister, J., Siddon, N. and Coupar, B. (2006)
CSIRO, Australia, Project report for the period January 2001 – December 2002
(Project ID No. 29495)

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About the report

This report has been prepared as part of the major review of Environment Australia Project 29495 “The Development of a Cane Toad Biological Control”. From January 2001 to December 2002, a joint research program was conducted between the CSIRO Divisions of Sustainable Ecosystems in Canberra and the Livestock Industries’ Australian Animal Health Laboratory in Geelong.

The long-term aim of the research is to develop a cane toad biological control by engineering an endemic, attenuated ranavirus, into which genes critical to the toad’s survival (i.e. present within the adult life stages) can be inserted. In the time frame of the current contract (2 years) the feasibility of the approach by conducting proof-of-concept research was undertaken. The report focuses primarily on the outcomes of the research against the project tasks and milestones detailed in the contract. Overall, the milestones have been met within the agreed time frames.

A breeding colony of the cane toad (Bufo marinus) has been successfully established and maintained in Canberra. Breeding techniques have been developed which enable the production of material for research on demand.

To attenuate the virus, two different approaches are being tried. Firstly, the virus has been subjected to multiple cell passages and secondly, genes in the virus have been identified that may attenuate the virus after their removal from the virus. The virus has been subjected to 100 passages and a deleted virus has been constructed. Both types of attenuated virus are currently being tested in an indicator species.

Work involving construction of a recombinant ranavirus has been highly successful. Insertion sites within the virus have been identified and a plasmid containing reporter and marker genes has been constructed. Protocols for the generation of recombinant viruses have been developed and the first recombinant ranavirus has been constructed. Attenuation testing of this virus is currently underway. Because of the highly methylated nature of ranavirus genomes, there was a high degree of uncertainty as to whether recombination could be achieved, and this success is a world first for this class of viruses.

The cane toad genes being targeted are those that are expressed in adults but not in tadpoles. The rationale for this approach is based on early work in the USA where bullfrog tadpoles died during metamorphosis if inoculated with adult haemoglobin; a gene product not expressed in the tadpole. It was concluded that the tadpole immune response to the adult haemoglobin compromised the tadpole’s passage through metamorphosis. To identify, isolate and express haemoglobin and other potential target genes, cDNA libraries have been constructed containing the coding sequences of different developmental stages of the cane toad. A “proof of principle” gene; adult ß-globin, has been cloned from the cane toad, expressed in vitro and purified. Antibodies against adult ß-globin were generated in rabbits. Inoculation trials were conducted where either the recombinant protein or the antibodies were inoculated into cane toad tadpoles. Analysis of the cane toads that arose from the protein injected tadpoles showed that inoculation of protein changes the expression of genes in the sub-adult. Analysis is currently underway of the antibody injected tadpoles. In addition, micro-array technology has been developed and used to identify more candidate genes.

Additional research was also conducted in order to develop more effective bio-control, both in the viral work and in the identification of genes. The report outlines this work. The report also discusses exciting and novel options for future research in developing a biological control for cane toads.