Suitability of canine herpesvirus as a vector for oral bait vaccination of foxes.

Studies were conducted to evaluate the feasibility of using canine herpesvirus (CHV) as a vaccine vector for bait-delivered oral vaccination of wild foxes. To test the viability of CHV in baits, CHV was freeze-dried, incorporated into different baits, stored, and the remaining viral infectivity tested in cell culture after varying periods of time at different storage temperatures. Experimental baits (mouse carcasses) and commercial baits (FOXOFF and PROBAIT) were prepared with either liquid or freeze-dried CHV and tested in two fox trials for their capacity to induce CHV-specific antibodies following oral baiting. Freeze-drying and storage temperatures below 0 degrees C had a stabilizing effect to virus infectivity. When stored at -20 degrees C, freeze-dried CHV retained its full infectivity for up to 3 months in PROBAIT baits, the remaining infectivity in FOXOFF baits was 100-fold less. Oral baiting with CHV induced antiviral serum antibodies in all vaccinated foxes (20/20). None of the vaccinated foxes became ill or shed infectious virus into the environment although viral DNA was detected in body secretions as evaluated by PCR. The results indicate that CHV can be freeze-dried and stored over extended periods of time without loosing much of its infectivity. This is the first report of CHV being used for oral bait vaccination of foxes. It appears that CHV is well suited for use as a recombinant vector for wild canids.

Development of canine herpesvirus based antifertility vaccines for foxes using bacterial artificial chromosomes.

Using bacterial artificial chromosome (BAC) technology, a canine herpesvirus (CHV)-based recombinant vaccine vector was produced for the development of an antifertility vaccine for foxes. Infectious viruses were recovered following transfection of canid cells with a BAC plasmid carrying the complete CHV genome. In vitro growth characteristics of BAC-derived viruses were similar to that of wildtype (wt)-CHV. Two recombinant antigens, fox zona pellucida protein subunit 3 (fZPC) and enhanced green fluorescent protein (EGFP) as control antigen, were inserted into thymidine kinase (TK) locus of the CHV genome and shown to be efficiently expressed in vitro. Inoculation of foxes with transgenic CHVs induced CHV specific antibodies, but was innocuous and failed to elicit transgene-specific antibody responses. Infectious virus or viral DNA was not detected in mucosal secretions or tissues of vaccinated foxes. The CHV-BAC system proved to be a quick and reliable method to manipulate the CHV genome. It will help to readily apply changes in the vector design in order to improve virus replication in vivo.

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.