Molecular contamination of an animal facility during and after African swine fever virus infection

African swine fever (ASF) is one of the most dangerous and devastating diseases of domestic pigs and wild boar. The disease poses a serious risk to the worldwide trade in pork meat, causing enormous economic losses. Since 2005, 74 countries have confirmed cases of ASF on their territory (29). The causative agent of ASF, African swine fever virus (ASFV), belongs to the Asfarviridae family. Its large genome (170 to 193 kilobase pairs), a double-stranded DNA, encodes more than 150 proteins (21). The virus has an ability to evade the host immune system, and causes massive, devastating inflammation processes leading to up to 100% mortality among affected animals (5, 26, 28). The complex structure of the virus and wide range of its genes prevent the development of an effective vaccine against ASF. Despite the promising results shown by several studies of vaccine candidates, currently there is no vaccine against the disease accepted as safe in the EU market (24, 31). Managing the disease relies on prevention by using strict biosecurity measures, early detection and culling of infected pigs. Such an approach has serious socio-economic impact (6, 13). Early restoring of pig production on farms affected by ASF could mitigate the economic losses; however, maximum safety should be observed in early restarting to prevent further disease spread.

According to the literature, ASFV is among the persistent pathogens in the environment, but the virus can be easily eradicated with most common disinfectants (10, 11, 14). Direct contact plays an important role in the transmission of the disease. Infected animals excrete the virus with oral and nasal fluids, faeces and urine. African swine fever virus can also be transmitted over short distances via aerosol. Contact with the blood of infected animals and their carcasses poses the highest risk of infection, as in those matrices the virus load is particularly large (27). The presence of blood (e.g. in bloody diarrhoea or after veterinary procedures) or any excreta in animal houses may result in contamination of the environment, and a consequent possibility of indirect transmission of ASF has been reported by several authors (18). On the one hand, field studies suggest that low farm biosecurity and contaminated fomites (e.g. clothes and vehicles) are the most probable facilitators of infection (17, 30). On the other hand, experimental infection of sentinel animals that had contact with contaminated environments could not be demonstrated at all (7) or was possible only very soon (i.e. one day) after removing infected pigs from the pigsty (19). A contaminated environment may pose a risk of ASF transmission because the virus persists in it. Davies et al. (4) proved that infectious ASFV could be found in faeces and urine for maximums of 15.33 and 3.71 days, respectively, depending on the temperature. Nuanualsuwan et al. (16) showed that at 25°C, infectious ASFV could be recovered from different fomites for no longer than 17 days (from non-porous materials) or 22 days (from porous materials). Therefore, the virus’ persistence as well as the characteristics of the individual farming systems affected by ASFV should be taken into consideration when biosecurity measures are implemented.

Legislation stipulates the procedure for introduction of a new pig herd onto a farm previously affected by ASF; in Poland it permits housing a new herd on a farm after 40 days, counting from the day when cleaning and disinfection processes are completed. Rehoused animals should serve as sentinels and be tested for the presence of specific anti-ASFV antibodies after 45 days. Shortly after the negative results are obtained, a new herd can be introduced safely. Alternatively, pigs may be brought onto the farm again 6 months from the completion of the cleaning and disinfection processes without retesting for antibodies (8). Such a long period ensures the safety of a newly introduced herd; however, in light of recently published studies in this area, from the economic point of view it may be open to discussion if this duration is necessary for the inactive phase (7, 19).

In the present study the molecular contamination of an animal facility was evaluated during and after highly pathogenic ASFV infection, to investigate the virus’ location and its initial amount in the environment. Isolation of infectious virus from collected samples was also attempted, to indicate the risk to animal safety from a contaminated environment. Different routes of transmission were focused upon, i.e. direct contact with oral and nasal excretions (the feeder, drinker and walls), contact with faeces (the drain, the floor and rubber floor mats), the aerosol route (the high-efficiency particulate-absorbing (HEPA) filter cover) and via personnel contact (doors).

After infection and replication in the host’s lymphatic organs, ASFV spreads through the pig’s body with blood and can soon be found in almost all tissues (1). The virus can be secreted with oral fluid, faeces and urine (4, 27). Shedding of the virus and the presence of dead animals in the pigsty worsen environmental contamination, pose a risk for indirect transmission of ASF and consequently block the sale of animals (2, 23). Presently, the only action to control the disease is the imposition of biosecurity measures (6). Knowledge of ASFV locations and virus load may facilitate cleaning and disinfection processes in the pigsty after culling, leading to effective eradication of the pathogen and fast reintroduction of animals onto a previously affected farm.

In this study, well-established shedding could be seen at 7 dpi. The presence of the virus’ DNA in all tested matrices is in accordance with previously published findings (9, 22, 25). The environmental samples collected in the present study reflected the ongoing infection situation and consequent facility contamination. Genetic traces of ASFV were found on surfaces that may have had contact with faeces and urine (the floor, walls and drain) and with oral and nasal excretions (the feeder and drinker). The latter outcome may suggest that the virus could be transmitted not only via surfaces but also via feed and water, which partially confirms other authors’ findings (3, 15, 18). The aerosol route was confirmed by finding traces of ASFV genetic material on the HEPA filter cover, which is in line with the observation of Olesen et al. (20) of proven infection of animals by this route over short distances.

In this study we were not able to isolate infectious virus from environmental samples. Attempts at infectious virus isolation directly from oral fluid were not successful either in the studies of Guinat et al. (9) or those of Davies et al. (4); however, the latter confirmed that infectious virus could be found in faeces and urine for up to approximately 5 days of the period of its shedding in similar environmental conditions (21°C). The results of our study may suggest that excretion of the virus to the environment renders it inactive, even when the initial virus load was high (as it was at the drain). These observations partially support those of the studies of Eblé et al. (7) and Olesen et al. (18), where infection of sentinel pigs was not possible at all or was possible no more than one day after removing ASF-affected animals from the facility. In light of this and previously published studies, the time needed for the introduction of a new pig herd onto a previously ASF-affected pig farm could be probably revised and shortened, on condition that similar environmental conditions are maintained.

The absence of genetic material of ASFV on doors indicates that using personal protective equipment (gloves) and disinfecting hands during standard veterinary procedures prevents further spreading of the virus. It was also noticeable that the estimated initial virus load decreased significantly on surfaces which had been cleaned mechanically with tap water. This showed that even simple maintenance of a pig-farming facility could minimise the risk of the pathogen’s transmission, but only on non-porous surfaces. Porous materials (i.e. in the present case, the rubber mats) were resistant to water washing and their estimated initial virus load did not decrease. This finding concurred with that of studies performed by Krug et al. (12) and Nuanualsuwan et al. (16), which indicated difficulties in effectively disinfecting porous materials.

Besides its use in proving the restoration of satisfactory hygiene standards, examination of environmental samples could be useful in epidemiological analysis, e.g. when ASF-affected pigs are transported illegally off a pig farm. Based on the results from this study, the presence of ASFV could be confirmed in the facility, especially in the places that had contact with sewage, such as the drain, but also on porous material and in the ventilation system.

To the best of our knowledge, this is the first report on contamination of a facility housing pigs during and after ASF infection. The gathered data provide useful practical knowledge, facilitating processes of cleaning and disinfection, and bring additional insight into the mechanism of indirect transmission of ASF and the virus presence in pig husbandry facilities.