ECTICONA

Contagious ecthyma (CE) is a viral disease caused by the of parapoxvirus. It affects ruminants and causes ulcers in facial mucus, nipples and hoofs and that can also affect humans. In less severe cases, the presence of lesions in animals makes eating difficult and affects the health condition and daily weight gain. There is a high incidence in the sheep livestock of Navarre, with considerable estimated economic losses derived from the infection itself and from healthcare costs. The costs are centred around acquiring antibiotics to alleviate secondary bacterial infections (mastitis and pododermatitis), because there is no treatment or vaccine for orf. Outbreaks start partly after acquiring animals from areas with a high prevalence. However, we currently do not have validated biotechnological tools that let us identify infected animals.

This project used ELISA tests developed in previous projects to do a sero-epidemiological study in Navarre. More than 1500 samples from 80 flocks were obtained, which we analysed and identified positive animals in 88% of them. A survey was done with all the ranchers at each livestock farm that provided evidence for the serious problem contagious ecthyma poses for flocks in Navarre and the need to develop vaccines.

Identifying infected animals is a fundamental strategy in implementing control programmes and keeping potentially dangerous animals from entering livestock farms. The project resulted in a field validation of an ELISA serological method for identifying animals infected with the orf virus with or without symptoms (figure 1).

The tool makes it possible to identify seropositive animals in the large majority of livestock farms analysed in a specific way (figure 2).

The presence of seropositive animals does not always coincide with outbreaks, and it could be related with prior vaccination campaigns that involved inoculation with the live virus that attenuate DIVA strategies, that differentiate infected animals from immunised ones, or make them impossible. In line with the surveys, up to 69% of the livestock farms have had outbreaks of the disease in the last five years, but only 55% of them were positive in ELISA. On the other hand, 21% of the livestock farms did not have outbreaks, but they had seropositive animals, probably from attenuated live virus vaccinations. In our territory, the disease predominantly appears in the mouth and mammary forms, which are two clinical forms that affect lamb yields and milk production, respectively.
As a second goal, the project developed viral vectors based on the Sendai (SeV) rat virus that immunogenic orf virus genes were inserted into. Specifically, two inoculants were used corresponding to the B2L and 0059 proteins encoded by the orf virus. Making use of the capability of SeV to infect the respiratory tract, its immunostimulant capacities, and the expression of a large amount of protein corresponding to the transgene, four groups of adult animals from a livestock farm without a history of orf or a serologic reaction to it were immunised.

• Control Group: inoculated with culture medium
• Recombinant Sendai (SeV-GFP) Group: inoculated with the Sendai vector that expresses the fluorescent green protein, without orf virus
• Group Immunised with B2L (SeV-B2L): inoculated with the Sendai vector that expresses the fluorescent green protein and gene B2L of the orf virus.
• Group Immunised with B2L (SeV-0059): inoculated with the Sendai vector that expresses the fluorescent green protein and gene 059 of the orf virus.

Identical intranasal immunizations and two experimental infections with orf virus isolated in the laboratory were done as part of the previous project. The animals in the control group developed the disease 45% of the time, and the ones inoculated with the recombinant vector only 18% of the time. On the other hand, the animals immunized with B2L showed illness 63% of the time, while the ones immunized with SeV-0059 did not show any sign of illness. Furthermore, only those animals showed a detectable level of orf antibodies in serum after the experimental infection (figure 3).

The ECTICONA (PC080) as a whole, alongside the previous ones (CONECTIM: PC071-072 and PC052-053), have made a contribution with the first genetic isolation and characterisation of strains of the orf virus, which causes CE, in our country. In addition, we showed the presence of antibodies in the blood of infected animals through the development of an ELISA that can recognise seropositive animals, and it has been applied to a large number of field samples, identifying a large majority of livestock farms with seropositive animals in Navarre. The importance of the disease for the livestock industry in Navarre has been made evident by results of the surveys, which depict CE as a disease that diminishes the productivity of sheep livestock and it highlights the need to develop tools that can be used to control it. The availability of the reverse genetic system of the Sendai virus made it possible to develop several recombinant viral vectors as vaccines for CE. The results in adult animals show the high potential of the SeV-059 vaccine to provide protection against experimental infection with the orf virus.

This project provided proof of concept for controlling CE by identifying seropositive animals with ELISA and protecting adult animals against the disease after experimental infection. The results should guarantee carrying out clinical trials in the most susceptible population (lambs), as well as gestating mothers, which would give us the opportunity to test the passive transference of immunity to the offspring. Thus, the ELISA and vaccine developed could make a definitive contribution to controlling the disease by using DIVA strategies and avoiding antibiotic use to treat infections, which is used in 60% of the cases in Navarre.