Assessment of virally vectored autoimmunity as a biocontrol strategy for cane toads.

BACKGROUND: The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner. METHODOLOGY/PRINCIPAL FINDINGS: The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs. CONCLUSIONS/SIGNIFICANCE: While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach.

Cane toad toxicity: an assessment of extracts from early developmental stages and adult tissues using MDCK cell culture.

Extracts of the cane toad (Bufo [Chaunus] marinus) adversely affected the growth of Mardin-Darby canine kidney (MDCK) cells during culture. In a similar manner to ouabain treatment, application of toad extracts over a 24 h period resulted in high levels of cytotoxicity, as indicated by cell detachment, increased membrane permeability and loss of mitochondrial function. Cell viability and growth were unchanged for controls (PBS) and increased with the application of Limnodynastes peronii tadpole and adult frog extracts. We investigated the general cytotoxicity of cane toad developmental stages (e.g., eggs, embryonic hatchlings, tadpoles and post-metamorphic toadlets) as well as selected adult tissues (e.g. skin, gut, liver). Our results showed that pre-metamorphic cane toad aqueous extracts used at 1 mg/ml on MDCK cells generated cytotoxicity levels comparable to ouabain treatment (3 microM). After normalisation, extracts from 2-3-month-old toadlets appeared less toxic than pre- and early metamorphic stages. Adult tissues revealed a gradient of cytotoxicity levels ranging from non-toxic brain to highly toxic dorsal skin extracts.

Experimental inoculation of European red foxes with recombinant vaccinia virus expressing zona pellucida C proteins.

The antifertility potential of zona pellucida proteins was tested in European red foxes by immunizing females with recombinant vaccinia viruses that express zona pellucida subunit C proteins. The fox zona pellucida C (fZPC) protein was newly identified and isolated as a cDNA clone from fox ovary RNA. Eighteen European foxes were inoculated with the recombinant vaccinia viruses or with wildtype vaccinia virus (wtVV) and their clinical, virological and immune responses evaluated. Following intradermal inoculation with wtVV or recombinant vaccinia virus expressing fox zona pellucida C (rVV-fZPC), or after peroral administration with recombinant vaccinia virus expressing the porcine zona pellucida C protein (rVV-pZPC) clinical signs of disease were not observed. Five out of six foxes developed antibodies to wtVV proteins. However, none of 12 foxes (six inoculated intradermally with rVV-fZPC, six perorally with rVV-pZPC) reacted in immunoblots with the transgenic fZPC or pZPC, respectively. Infectious wtVV, rVV-fZPC or rVV-pZPC was not isolated from mucosal secretions of any of the foxes whereas viral DNA was present in oral swabs of 3/18 foxes as determined by PCR. Hematological parameters remained mostly unchanged. Histopathological changes were not observed in the ovaries of rVV-fZPC or wtVV inoculated foxes. The data indicate that inoculation of foxes with cell culture infectious wtVV, rVV-fZPC or rVV-pZPC did not result in production of infectious progeny virus in vivo. For this reason transgene expression may have been insufficient to induce adequate immune responses against the transgenic proteins.

Corticosteroid treatment does not reactivate canine herpesvirus in red foxes.

To study canine herpesvirus (CHV) reactivation from red foxes (Vulpes vulpes), 29 foxes with varying CHV antibody and CHV carrier status were treated with methylprednisolone acetate, a glucocorticosteroid drug with prolonged immunosuppressive effect in dogs. In the first experiment, 17 foxes with unknown CHV carrier status were treated once with methylprednisolone: in the second experiment, five foxes were treated twice, 4 mo after being intravenously CHV infected; and in the third experiment, six foxes were treated five times, 11 mo after peroral CHV infection. Infectious CHV was not isolated after treatment from either naturally or experimentally CHV-infected foxes or from untreated, CHV-seronegative in-contact foxes. Canine herpesvirus DNA was not detectable in mucosal secretions or white blood cells of any of the foxes, whereas all trigeminal ganglia of experimentally CHV-infected foxes were polymerase chain reaction-positive. In CHV-seropositive foxes, anti-CHV antibody titers did not change with time after treatment, and CHV-seronegative in-contact controls did not seroconvert. Hematologic parameters remained mostly unchanged. We conclude that CHV is not as easily reactivated in foxes following corticosteroid treatment as in dogs, although there was no obvious sign of immunosuppression. Canine herpesvirus was not spread from virus carriers to naive in-contact foxes, which may be among possible explanations for the reported low CHV prevalence in wild foxes.