Discussion
African swine fever is no longer an exotic disease and has established self-sustaining, complicated transmission cycles in European wild boar populations. Slow but constant local spread is observed (data from the Animal Disease Notification System, visited online May 8th, 2020). This was rather unexpected as historical experience did not indicate that wild boar could sustain an endemic infection cycle (Laddomada et al., 1994). Field observations and experimental studies indicate a high lethality (Blome et al., 2012, Gabriel et al., 2011) and low contagiosity, especially in the initial phase of an ASF outbreak among wild boar. The low level of contagiosity requires a rethinking and an adapted approach to control ASF in the wild boar population (Depner et al., 2016a, Depner et al., 2016b). Evidence suggests that ASF in a feral pig population tends to behave more like a long-term (rather stationary) habitat-bound disease with no tendency to spread rapidly. It is mainly infectious cadavers, combined with the high tenacity of the ASF virus and the low contagiosity, that can contain the disease within a region (Depner et al., 2016b). ASFV-contaminated soil rooted by wild boar is one of the habitat factors that could play a role in transmission. Probst et al. (2017) reported that wild boar show interest in the soil where carcasses have been found previously, with wildlife cameras documenting animals rooting in soil even when only bones remained. Furthermore, Estonian colleagues and others have demonstrated viral genome in these soils (Viltrop and Nurmoja, personal communication)(Zani et al., 2020).
In our study we tried to create a data set for a risk assessment of the role of contaminated soil in ASFV transmission and possible mitigation measures.
We demonstrate that virus stability depends on the soil type, pH, organic material percentage, and to a lesser extent, on ambient temperature. While contaminated sand retains infectivity for weeks, virus stability is very low in acidic forest soils. Intermediate times were found in swamp mud and yard soil. Within the limits of our experimental setup, and assuming that the animal is an even more sensitive detection system, we cannot rule out a persistence of infectivity for at least a couple of weeks. The residual infectivity was within the range that was shown to be infectious when orally applied to susceptible animals (McVicar, 1984, Pietschmann et al., 2015). These results contradict to a certain extent previously published studies (Mazur-Panasiuk and Wozniakowski, 2020), where water, soil and leaf litter inactivated ASFV quickly. In this study Mazur-Panasiuk and Wozniakowski (2020) were able to re-isolate ASFV from soil and leaf litter immediately after adding culture supernatant to the matrix, but even a short 3-day incubation caused complete loss of virus infectivity independent of temperature conditions. This is in line with our results from swamp mud but not from yard soil or sand, where much longer periods of infectivity were observed. In contrast, re-isolation immediately after adding the contaminant to forest soil was impossible in our hands. Thus, virus inactivation seems to occur after short contact with the matrix e.g. due to the acidic conditions in both forest soil specimens (pH 4.1 and 3.2).
Risk mitigation could involve the use of disinfectants despite the obvious limitation that decontamination of soils in fields and forests, which are very different in structure, consistency and composition, is generally difficult and the organic matter in body fluids impairs disinfection (Weber et al., 1999). We used citric acid and calcium hydroxide in our study, which both have proven efficiency against ASFV (Turner and Williams, 1999, Krug et al., 2012), the former with known inhibition by blood (Krug et al., 2018). It must be noted that in the past, lime products were used in the control of classical swine fever in wild boar, e.g. in Germany. It is assumed that they not only have a disinfectant effect but also repel wild boar. Furthermore, these products were well accepted by the hunters. The application of lime was therefore included in the official recommendation of the German government for the use of disinfectants in an epizootic (Blome et al., 2020). Despite the above information, it can be questioned whether the application of a basic chemical to acidic soils in the wild boar habitat is appropriate. ASFV is quite reliably inactivated at a pH of below 4 (EFSA, , 2009). Therefore, acidic disinfectants could be more useful and here, citric acid was our candidate.
In our study, ASFV was inactivated after 1 h of disinfectant treatment. In spiked beach sand and commercial potting soil not treated with disinfectant, ASFV was fully inactivated after 2 weeks. However, untreated blood or sterile sand were infectious for the entire test interval of 3 weeks with consistent results from virus isolation (macrophages) and titration on WSL cells.
In conclusion, ASFV stability is very low in forest soils but rather high in sandy soils. Given the high variability of wild boar habitats and unforeseen effects of the decay matrix, treatment of carcass locations with disinfectants should be considered when setting up control measures. The powder format of the used chemicals could be beneficial and practical. Nevertheless, regulations on the use of biocides and occupational safety have to be considered. Off-label use of commercial products could be an alternative. In this context, disinfectants based on potassium peroxymonosulfate (Trifectant, Virkon S) were recently shown to inactivate ASFV on porous surfaces (Gabbert et al., 2020) but had problems with blood under certain circumstances (Krug et al., 2018). Removal of ASFV-positive carcasses is of utmost importance and remains a critical control measure as live virus may remain infectious in certain soil matrices for weeks. These studies establish useful protocols to isolate ASFV from soil matrices, while providing insight to potential management options useful in the field to mitigate transmission.