Parapoxviruses (PPVs) (family Poxviridae) cause dermal diseases most commonly in sheep, goats and cattle 2, but also in semi-domesticated reindeer (Rangifer tarandus tarandus) 3 and wildlife, like seals 4, red deer 5 and squirrels 6. The genus Parapoxvirus consists of five species and three tentative species 7: Bovine popular stomatitis virus (BPSV), orf virus (ORFV), parapoxvirus of red deer in New Zealand (PVNZ), pseudocowpox virus (PCPV), and squirrel parapoxvirus (SPPV), as well as the tentative species of the genus: auzdyk disease virus, chamois contagious ecthyma virus and sealpox virus.

The first documented outbreak of a contagious ecthyma-like disease in Norwegian reindeer, took place in 1976 among experimental animals at the National Reindeer Research Station in Lødingen 8. Also human parapoxvirus infections were associated with this outbreak 9. In April 1999, the first outbreak in reindeer under regular herding conditions was reported in Troms County, followed by an outbreak one year later, also in April, in Nordland County. The outbreak in 2000 involved at least 30 out of 150 reindeer, which were corralled and supplementary fed during the winter. Seven of the infected animals died, due to massive oral cauliflower-like lesions and secondary bacterial infections 1.

In Norway, there are both wild and semi-domesticated reindeer. The latter is herded over an area of approximately 140.000 km², which is about 40% of the mainland area of Norway. Most reindeer herding is conducted by the Saami people (indigenous people of Scandinavia), and the most dense reindeer herding area is in Finnmark county, Northern Norway, where the total number of animals is estimated to be around 140.000 animals (estimate for 2003 10). Semi-domesticated reindeer have seasonal migrations between winter (usually inland) and summer (usually coastal areas) pastures, and the pastures are organised in districts, reducing the contact between animals of different districts. However, sheep and goats also use the coastal pastures during spring, summer and autumn, and contact between different animal species is possible. Furthermore, it is not uncommon to use the same corrals and transport vehicles for reindeer and sheep, and remaining scabs from sheep with contagious ecthyma may as well be a source of infection for reindeer. Since the disease contagious ecthyma and orf virus is present among sheep and goats in most parts of the country, it is necessary to characterise the causative agent of contagious ecthyma in reindeer, in order to find out whether reindeer have their own parapoxvirus species, like red deer in New Zealand 5, or whether a transmission of virus between sheep and goats and reindeer is more likely.

Due to genetic heterogeneity within the genus, classification of PPV remains problematic and relies mostly on the source of the virus isolate; id est Parapoxviruses isolated from sheep will be classified as orf virus, virus isolated from cattle as pseudocowpox 1112. However, several molecular techniques have been used to characterise parapoxviruses. Inoshima et al. 13 reported a polymerase chain reaction (PCR) protocol assumed to be able to amplify all members of the parapoxvirus genus, based on primers from the B2L gene encoding a major envelope protein 14. The gene region encoding a viral interleukin 10 orthologue (vIL-10) 15 has been used for characterisation, in combination with sequencing of the amplified DNA products. Restriction fragment length polymorphism (RFLP) has also been used to compare parapoxvirus isolates 516, and can be conducted both on genomic DNA as well as on smaller DNA fragments in combination with PCR. Also random amplified polymorphic DNA analysis (RAPD) may be used for characterisation purposes, as reported as a useful technique for comparing orthopoxviruses 17.

The aim of the present study was to characterise the causative agent of contagious ecthyma in semi-domesticated reindeer in Norway, and to compare this virus with other parapoxvirus isolates. This information is necessary to be able to sort out whether reindeer are exposed to orf virus, which is commonly affecting sheep and goats and also present among muskoxen in Norway, to bovine papillar stomatitis virus, which is present among cattle in Norway, or whether reindeer hosts a specific parapoxvirus species.

Based on RFLP patterns obtained from the Norwegian reindeer isolates and orf NZ2, a close relationship between these viruses can be assumed (Figure 1). The RAPD patterns of the reindeer isolates and the standard orf virus strains orf 11 and orf NZ2 show high similarity in amplification patterns (Figure 2). This similarity is present in spite of the geographical distance between the Norwegian isolates and the orf strains originating from Great Britain and New Zealand.

Further, this work demonstrates that a virus species characterisation based on the nucleotide sequence of the PCR-product from the B2L-PCR is possible. This characterisation method is easy to perform, because the PCR product is fast to obtain and to proceed.

The phylogenetic analysis of the partial B2L-gene sequence from the Norwegian reindeer isolates shows that these isolates can be allocated to the orf virus species. However, the clade credibility value of 37 % also demonstrate a close relationship between the two different parapoxvirus species, orf and pseudocowpox virus.

Tikkanen et al. 18 demonstrated the affiliation of parapoxvirus isolates obtained from Finnish semi-domesticated reindeer during the early outbreaks (1992–1994) in Finland, to the orf virus species and from later outbreaks to the pseudocowpox viruses, which is congruent with our results.

The close clustering of the translated sequences obtained from virus isolated from reindeer (two Norwegian; 1999, 2000, and one Finnish; 1994) with the amino acid sequences of sheep and goat interleukin 10 demonstrate a high relationship of the parapoxvirus isolated from reindeer to the main hosts of orf virus (sheep and goat). It seems that the viral interleukin 10 orthologue of the reindeer isolates is highly adapted to the immune system of sheep and goats, which also indicates that the reindeer parapoxvirus belongs to the orf virus species.

Our results indicate that neither geographical distance, nor crossing the host barrier from sheep or goat to semi-domesticated reindeer did affect the characteristics of the parapoxvirus orf virus investigated in this study.

The B2L PCR has previously been described as a tool to amplify all species within the parapoxvirus genus 13. However, we were not able to amplify B2L sequences from nine of the twenty-five isolates included in our study. Many members of the subfamily chordopoxvirinae show genetic rearrangement at the terminal sequences of the genome, which is thought to be an evolutionary mechanism, allowing the virus to adapt to changes of the immune response of the host 19. Parapoxvirus replicated in vitro show rearrangement of the left terminal end of the viral genome, resulting in the deletion of three genes (E2L, E3L, G1L) and 80 % of a fourth one, the G2L 202122. All these four genes play a major role in virulence and host specificity 23. The B2L gene is localised beside the left terminal end of the genome, so that heterogeneity in the primer binding regions or complete deletion of the B2L gene, through the same rearrangement mechanisms as in the left terminal region itself, can be the reason for the negative results of the B2L-PCR. However, further analyses are needed to evaluate this hypothesis. As compared to the results of the B2L-PCR, the GIF PCR seems to be more sensitive for different members of the genus and can be used for rapid genus-identification. However, the GIF gene is localised in the right terminal gene region 24 and may also undergo genetic rearrangements due to adaptation processes. The GIF gene has so far only been detected in parapoxviruses 23.

The vIL-10-PCR amplified all tested isolates, but rearrangements of the vIL-10 gene have also been demonstrated, in terms of duplications in the inverted terminal repeat 25. Thus, genetic rearrangements may be a problem when designing a PCR that are supposed to detect parapoxviruses in general. The use of a multiplex PCR, with a combination of one or more of the gene targets desribed above, may thus be a solution to this problem.

The results of this work point out that the parapoxvirus that has caused contagious ecthyma in Norwegian semi-domesticated reindeer belongs to the orf virus species, and it is to assume that the orf virus crosses the host species barrier from sheep and goat to semi-domesticated reindeer.

Sheep and goats in Norway are commonly free-ranging during the snow-free period, sharing pastures with semi-domesticated reindeer. During seasonal migrations and corralling of animals for slaughter etc. they may also share fences, transport vehicles and other equipment.

As far as we know, contagious ecthyma is the single disease that has caused the most serious economical losses for reindeer herders in the Nordic countries in recent times, and especially so in Finland. Consequently, the common use of equipment, pastures, transport vehicles and facilities for semi-domesticated reindeer, sheep and goats should be avoided, to prevent cross infections.

The outcome of parapoxvirus infections in reindeer seems to be dependent on many environmental factors in addition to the exposure to the virus. Certain types of stress is believed to play a key role, and such stress factors may be lack of food, as well as handling, corralling and transport of animals 31. A changing trend in reindeer herding conditions, facing higher animal densities, faster movements of animals using helicopter and snow mobiles, increased use of supplemental feeding and corralling of animals, and increased transport distances to slaughterhouse may represent factors that can predispose for contagious ecthyma as well as other diseases in reindeer.

The author(s) declare that they have no competing interests.

This work is based on the MPhil thesis of Jörn Klein. Morten Tryland was the main supervisor of this thesis and project leader. The thesis is available online under: http://uit.no/getfile.php?PageId=647&FileId=26