J Vet Res 67, 503-508, 2023 DOI: 10.2478/jvetres-2023-0065 Molecular contamination of an animal facility during and after African swine fever virus infection Marek Walczak1, Krzesimir Szymankiewicz1, Fernando Rodriguez2,3,4, Jordi Argilaguet2,3,4, Boris Gavrilov5, Jacek Żmudzki1, Maciej Kochanowski1, Małgorzata Juszkiewicz1, Anna Szczotka-Bochniarz1 1Department of Swine Diseases, National Veterinary Research Institute, 24-100 Puławy, Poland 2 Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain 3IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain 4WOAH Collaborating Centre for Emerging and Re-emerging Pig Diseases in Europe, IRTA-CReSA, 08193 Barcelona, Spain 5Biologics Development, Huvepharma, 1113 Sofia, Bulgaria marek.walczak@piwet.pulawy.pl Received: July 18, 2023 Accepted: November 14, 2023 Abstract Introduction: The molecular contamination of an animal facility was investigated during and after an infection with highly pathogenic African swine fever virus (ASFV) among domestic pigs. The investigation evaluated the risk of indirect transmission of the disease and indicated points that may facilitate cleaning and disinfection processes. Material and Methods: Six domestic pigs were infected oronasally with the highly pathogenic Georgia 2007 strain. Environmental samples from the floors, walls, rubber floor mats, feeders, drinkers, high-efficiency particulate-absorbing filter covers and doors were collected 7 days post infection (dpi), 7 days later and 24 h after disinfection of the facility. The samples were investigated by real-time PCR and in vitro assays to find genetic traces of ASFV and infectious virus. Results: Typical clinical outcomes for ASF (i.e. fever, apathy, recumbency and bloody diarrhoea) were observed, and all animals died or required euthanasia before or at 9 dpi. No infectious virus was found in environmental samples at the sampling time points. Genetic traces of ASFV were found in all locations except the doors. The initial virus load was calculated using real-time PCR threshold cycle values and was the highest at the drain. A statistically significant decrease of virus load over time was found on non-porous surfaces mechanically cleaned by water (the floor and drain). Conclusion: The gathered data confirmed different routes of virus excretion (oral and nasal, faeces and urine, and aerosol) and showed virus locations and different initial concentrations in the animal facility. Maintaining the facility with mechanical cleaning and using personal protection (gloves) and hand disinfection may efficiently minimise the risk of further virus spread. Together with the results of previously published studies, the present investigations’ failure to isolate infectious virus may suggest that if stable environmental conditions are assured, the time needed before the introduction of new herds into previously ASF-affected farm facilities could be shortened and in this way the economic losses caused by the disease outbreak mitigated. Keywords: African swine fever, pigsty, indirect transmission, persistence, risk. Introduction causative agent of ASF, African swine fever virus (ASFV), belongs to the Asfarviridae family. Its large African swine fever (ASF) is one of the most genome (170 to 193 kilobase pairs), a double-stranded dangerous and devastating diseases of domestic pigs and DNA, encodes more than 150 proteins (21). The virus wild boar. The disease poses a serious risk to the has an ability to evade the host immune system, and worldwide trade in pork meat, causing enormous causes massive, devastating inflammation processes economic losses. Since 2005, 74 countries have leading to up to 100% mortality among affected animals confirmed cases of ASF on their territory (29). The (5, 26, 28). The complex structure of the virus and wide © 2023 M. Walczak et al. This is an open access article distributed under the Creative Commons Attribution- NonCommercial-NoDerivs license (http://creativecommons.org/licenses/by-nc-nd/3.0/) 504 M. Walczak et al./J Vet Res/67 (2023) 503-508 range of its genes prevent the development of antibodies (8). Such a long period ensures the safety of an effective vaccine against ASF. Despite the promising a newly introduced herd; however, in light of recently results shown by several studies of vaccine candidates, published studies in this area, from the economic point currently there is no vaccine against the disease accepted of view it may be open to discussion if this duration is as safe in the EU market (24, 31). Managing the disease necessary for the inactive phase (7, 19). relies on prevention by using strict biosecurity measures, In the present study the molecular contamination of early detection and culling of infected pigs. Such an animal facility was evaluated during and after highly an approach has serious socio-economic impact (6, 13). pathogenic ASFV infection, to investigate the virus’ Early restoring of pig production on farms affected by location and its initial amount in the environment. ASF could mitigate the economic losses; however, Isolation of infectious virus from collected samples was maximum safety should be observed in early restarting also attempted, to indicate the risk to animal safety to prevent further disease spread. from a contaminated environment. Different routes of According to the literature, ASFV is among the transmission were focused upon, i.e. direct contact with persistent pathogens in the environment, but the virus oral and nasal excretions (the feeder, drinker and walls), can be easily eradicated with most common disinfectants contact with faeces (the drain, the floor and rubber floor (10, 11, 14). Direct contact plays an important role in the mats), the aerosol route (the high-efficiency particulate- transmission of the disease. Infected animals excrete the absorbing (HEPA) filter cover) and via personnel virus with oral and nasal fluids, faeces and urine. African contact (doors). 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 Material and Methods infection, as in those matrices the virus load is particularly large (27). The presence of blood (e.g. in Animals. The data presented in this study were bloody diarrhoea or after veterinary procedures) or any gathered during an independent animal trial from excreta in animal houses may result in contamination of a group of six 10-week-old Danbred Duroc domestic the environment, and a consequent possibility of indirect pigs. The animal experiment was approved by the Local transmission of ASF has been reported by several Ethical Committee for Animal Experiments in Lublin authors (18). On the one hand, field studies suggest that (under approval number 82/2022). All procedures low farm biosecurity and contaminated fomites including euthanasia were performed in compliance (e.g. clothes and vehicles) are the most probable with current legal regulations. facilitators of infection (17, 30). On the other hand, Virus. The virulent ASFV genotype II Georgia experimental infection of sentinel animals that had 2007 strain at a dose of 1 × 105 50% haemadsorbing contact with contaminated environments could not be doses (HAD50) per animal was used for intranasal demonstrated at all (7) or was possible only very soon infection. The virus was isolated by Dr. Linda Dixon (i.e. one day) after removing infected pigs from the at the Pirbright Institute, Woking, UK, and kindly pigsty (19). A contaminated environment may pose provided for the present study by the Institut de Recerca a risk of ASF transmission because the virus persists in it. i Tecnologia Agroalimentàries – Centre de Recerca Davies et al. (4) proved that infectious ASFV could be en Sanitat Animal, Barcelona, Spain. found in faeces and urine for maximums of 15.33 and Facility. Animals were kept in a biosafety level 3 3.71 days, respectively, depending on the temperature. animal facility at the National Veterinary Research Nuanualsuwan et al. (16) showed that at 25°C, Institute (Puławy, Poland) and provided with feed and infectious ASFV could be recovered from different water ad libitum. Before the experiment, the state of fomites for no longer than 17 days (from non-porous health of all the animals was evaluated through materials) or 22 days (from porous materials). veterinary examination, and the six pigs were confirmed Therefore, the virus’ persistence as well as the to be free of ASFV by testing with a Virotype Real-time characteristics of the individual farming systems PCR kit (Qiagen, Hilden, Germany). The facility affected by ASFV should be taken into consideration maintains a stable temperature ranging from 20 to 24°C, when biosecurity measures are implemented. humidity at 55% (±10%), 12–15 air exchange per hour Legislation stipulates the procedure for introduction and airflow not exceeding 0.3 m/s. Pigs were kept of a new pig herd onto a farm previously affected by without other bedding than rubber mats ensuring their ASF; in Poland it permits housing a new herd on a farm welfare. after 40 days, counting from the day when cleaning and Cleaning and disinfection. The facility floor was disinfection processes are completed. Rehoused animals cleaned with tap water on a daily basis during the trial should serve as sentinels and be tested for the presence by the facility staff. Seven days from the end of the of specific anti-ASFV antibodies after 45 days. Shortly experiment, the facility was decontaminated with 35% after the negative results are obtained, a new herd can be hydrogen peroxide vaporisation (Bioquell Z, Andover, introduced safely. Alternatively, pigs may be brought UK). onto the farm again 6 months from the completion of the Shedding assessment. Oral, nasal and rectal swabs cleaning and disinfection processes without retesting for were collected at 0 and 7 days post infection (dpi). M. Walczak et al./J Vet Res/67 (2023) 503-508 505 Additional samples were taken during necropsy. Swabs way. The plates were incubated at 37°C in 5% CO2 and were placed into tubes containing 1 mL of phosphate- inspected for the presence of haemadsorption and buffered saline, then incubated at room temperature for cytopathic effect on the 0, 4th and 7th days of incubation. 10 min and vortexed. An aliquot of 200 µL of each In addition, cells and medium were collected on days 0 sample was reserved for DNA extraction and real-time and 7 of incubation for real-time PCR analyses after PCR analysis. three freeze-thaw cycles. Environmental sample collection. Environmental Initial virus load estimation. The initial virus load samples were collected when the infection was ongoing was estimated for non-infectious samples based on the at 7 dpi, at 14 dpi (before decontamination) and at 15 dpi threshold cycle (Ct) values, as previously described (27). (24 h after decontamination). Two millilitres of Roswell Statistical analysis. Statistical analysis of initial Park Memorial Institute 1640 medium (PAN Biotech, virus load in samples from different time points was Aidenbach, Germany) as a growth medium (GM) performed using the Kruskal–Wallis test in GraphPad supplemented by 1% Antibiotic-Antimycotic Solution Prism 8.4.2 (GraphPad Software, San Diego, CA, USA). (Sigma-Aldrich, St. Louis, MO, USA) and 10% foetal The significance for statistical difference was defined as bovine serum (GIBCO, Thermo Fisher Scientific, P-value < 0.05. Waltham, MA, USA) was placed into a plastic tube. Designated spots were thoroughly scrubbed by swabs and immersed in the medium. Swabs from the floor (five Results spots), feeder (one spot), drinker (one spot), a rubber mat (one spot), a wall at a height of about 30 cm (one spot), Infection outcomes and virus shedding. Fever the HEPA filter cover (one spot), and the doors followed by apathy in infected animals could be seen at a height of about 120 cm (one spot) were collected in beginning from 4 dpi. Infection developed and resulted triplicate. Samples were aliquoted and stored at −80°C in the deaths of 100% of the animals by 9 dpi. During for real-time PCR and in vitro analyses. the disease, three out of the six animals reached the DNA extraction and real-time PCR. Extraction humane endpoint and were euthanised at 8 dpi because of DNA was carried out on the Qiagen DNA Mini Kit they were suffering from bloody diarrhoea, severe protocol (Qiagen), with further use of the Virotype dyspnoea and recumbency. The evolution of fever and ASFV PCR Kit (Qiagen) for a subsequent real-time PCR the survival rate in this group are presented in Fig. 1. reaction in a Rotor-Gene Q thermocycler (Qiagen) Up to the 7th dpi (the first environmental sampling according to the manufacturer’s instructions. time point), four out of the six animals remained in the Infectious virus isolation. Porcine primary facility, and at this time, the presence of ASFV genetic pulmonary alveolar macrophages were collected by lung material was confirmed in the rectal swabs of two out of lavage from uninfected donor pigs and seeded at 1 × 105 the four animals and the oral and nasal swabs of all four cells per well in GM supplemented by erythrocytes animals. All swab samples collected from all six animals (1 : 300 v/v) in a 96-well plate. One hundred microlitres during necropsy were PCR positive, with noticeably of each filtered (0.45 µm) environmental sample was higher initial virus loads. The mean virus loads of swab added to the respective wells in triplicate. In parallel, the samples collected at 7 dpi and during necropsy based on negative control (medium) and positive control (Georgia the Ct values is presented in Fig. 2. 2007, 1 × 103 HAD50/mL) were prepared in the same Fig. 1. Survival rate and fever evolution during experimentally induced African swine fever in 10-week-old Danbred Duroc pigs. Dots indicate environmental sampling timepoints. Red dots – before disinfection; green dot – after disinfection; dashed black line – fever threshold; dpi – days post infection 506 M. Walczak et al./J Vet Res/67 (2023) 503-508 Table 1. Numbers of positive qPCR-samples in tested animal facility locations and their mean threshold cycle (Ct) values Number of positive samples Time (Mean Ct (±SD)) Floor Wall Door HEPA filter cover Drain Feeder Drinker Mat 14/15 3/3 2/3 2/3 3/3 3/3 2/3 T1 0/3 (35.9 (±1.6)) (33.3 (±1.9)) (37.3 (±0.4)) (28.7 (±1.4)) (34.3 (±1.9)) (33.0 (±0.8)) (38.3 (±0.6)) 4/15 2/3 1/3 1/3 3/3 3/3 3/3 T2 0/3 (38.2 (±0.6)) (36.7 (±2.2)) (36.7 (±0.0)) (32.4 (±0.2)) (35.9 (±1.2)) (36.7 (±1.6) (37.8 (±0.9)) 5/15 2/3 1/3 1/3 3/3 3/3 3/3 T3 0/3 (39.0 (±0.8)) (35.6 (±2.9)) (39.1 (±0.0)) (32.9 (±0.6)) (36.2 (±1.6)) (34.6 (±1.6)) (37.4 (±0.7)) SD – standard deviation; T1 – ongoing infection (7 days post infection (dpi)); T2 – after infection (T1 + 7, i.e. 14 dpi), T3 – 24 h after disinfection (15 dpi); HEPA – high-efficiency particulate-absorbing The highest estimated initial virus load was found while the infection was ongoing (7 dpi) at the drain (reaching 1.92 × 104 eqTCID50/mL). A noticeably decreasing tendency in estimated virus load over time was found at the floor and drain spots at 15 dpi, when the mean virus load had decreased to 5.5 × 101 50% tissue culture infectious dose equivalents (eqTCID50)/mL and 1.2 × 103 eqTCID50/mL, respectively. These decreases were statistically significant changes, which was shown by a P-value of 0.002 for the floor and one of 0.0393 for the drain. It is noteworthy that the mean initial virus load at feeder and drinker spots was slightly higher than at faeces and urine or aerosol contact spots (i.e. the mats, the floor and the HEPA filter cover). The estimated initial virus loads in different locations and their change over time is presented in Fig. 3. Fig. 2. Estimated initial virus load in swab samples collected from 10-week-old Danbred Duroc pigs during experimentally induced African swine fever at 7 days post infection (dpi) and necropsy. The Discussion boxes represent the 50% between the 25 and 75% quartiles. The line inside the box indicates the median. The whiskers denote maximum and minimum values. eqTCID50 – 50% tissue culture infectious dose After infection and replication in the host’s lymphatic equivalent 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. Fig. 3. Initial load of African swine fever virus in different pigsty locations after experimental infection and its change over time. In this study, well-established shedding could be Statistically significant decreases of estimated initial virus load were seen at 7 dpi. The presence of the virus’ DNA in all observed at 15 dpi at the floor and drain spots – locations mechanically tested matrices is in accordance with previously cleaned by water. Error bars indicate standard deviation. eqTCID50 – published findings (9, 22, 25). The environmental 50% tissue culture infectious dose equivalent; HEPA – high-efficiency particulate-absorbing; * – statistically significant (P-value 0.002 samples collected in the present study reflected the (floor), 0.0393 (drain)) ongoing infection situation and consequent facility contamination. Genetic traces of ASFV were found on Environmental samples. The presence of ASFV surfaces that may have had contact with faeces and urine DNA could be confirmed in all tested locations except (the floor, walls and drain) and with oral and nasal the doors. However, infectious virus could not be excretions (the feeder and drinker). The latter outcome recovered in any of the samples collected at any time may suggest that the virus could be transmitted not only point. The numbers of qPCR-positive samples at the via surfaces but also via feed and water, which partially three sampling time points are presented in Table 1. confirms other authors’ findings (3, 15, 18). The aerosol M. Walczak et al./J Vet Res/67 (2023) 503-508 507 route was confirmed by finding traces of ASFV genetic Conflicts of Interests Statement: The authors declare material on the HEPA filter cover, which is in line with that there is no conflict of interests regarding the the observation of Olesen et al. (20) of proven infection publication of this article. of animals by this route over short distances. In this study we were not able to isolate infectious Financial Disclosure Statement: This study received virus from environmental samples. Attempts at no external funding. infectious virus isolation directly from oral fluid were not successful either in the studies of Guinat et al. (9) or Animal Rights Statement: The experiment was those of Davies et al. (4); however, the latter confirmed approved by the Local Ethical Committee for Animal that infectious virus could be found in faeces and urine Experiments in Lublin (approval number 82/2022). for up to approximately 5 days of the period of its All procedures were carried out according to the “Ustawa shedding in similar environmental conditions (21°C). z dnia 15 stycznia 2015 r. o ochronie zwierząt The results of our study may suggest that excretion of wykorzystywanych do celów naukowych lub edukacyjnych the virus to the environment renders it inactive, even (Dz. U. z 2015, poz 266)” (Polish Act of 15 January when the initial virus load was high (as it was at the 2015 on the protection of animals used for scientific or drain). These observations partially support those of the educational purposes (Official Journal of Laws 2015, studies of Eblé et al. (7) and Olesen et al. (18), where item 266) based on Directive 2010/63/EU of the infection of sentinel pigs was not possible at all or was European Parliament and of the Council of 22 September possible no more than one day after removing 2010 on the protection of animals used for scientific ASF-affected animals from the facility. In light of this purposes. and previously published studies, the time needed for the introduction of a new pig herd onto a previously Acknowledgements: We are grateful to Dr. Linda ASF-affected pig farm could be probably revised and Dixon for providing the virus strain for this research. We shortened, on condition that similar environmental thank María Jesús Navas and David Marín for their conditions are maintained. excellent support during this study. The absence of genetic material of ASFV on doors indicates that using personal protective equipment (gloves) and disinfecting hands during standard veterinary References procedures prevents further spreading of the virus. 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