Short time window for transmissibility of African swine fever virus from a contaminated environment

Since the introduction of African swine fever virus (ASFV ) into the Baltic states and Poland in 2014, the disease has continued to spread within these regions. In 2017, the virus spread further west and the first cases of disease were reported in the Czech Republic and Romania, in wild boar and domestic pigs, respectively. To control further spread, knowledge of different modes of transmission, including indirect transmission via a contaminated environment, is crucial. Up until now, such an indirect mode of transmission has not been demonstrated. In this study, transmission via an environment contaminated with excretions from ASFV ‐infected pigs was investigated. Following euthanasia of pigs that were infected with an isolate of ASFV from Poland (POL /2015/Podlaskie/Lindholm), healthy pigs were introduced into the pens, in which the ASFV ‐infected pigs had been housed. Introduction was performed at 1, 3, 5 or 7 days, following euthanasia of the infected pig groups. Pigs, that were introduced into the contaminated environment after 1 day, developed clinical disease within 1 week, and both ASFV DNA and infectious virus were isolated from their blood. However, pigs introduced into the contaminated pens after 3, 5 or 7 days did not develop any signs of ASFV infection and no viral DNA was detected in blood samples obtained from these pigs within the following 3 weeks. Thus, it was shown that exposure of pigs to an environment contaminated with ASFV can result in infection. However, the time window for transmissibility of ASFV seems very limited, and, within our experimental system, there appears to be a rapid decrease in the infectivity of ASFV in the environment.     

Detection of African swine fever virus in feed dust collected from experimentally inoculated complete feed using quantitative PCR and virus titration assays

African swine fever virus (ASFV) is a current threat to global pork production due to its high case fatality rate, lack of efficacious vaccine and recent transboundary spread into new regions of the world. Preventing introduction and further spread of ASFV is critical for countries currently negative for the virus. ASFV is stable in feed ingredients subjected to transoceanic conditions and transmission occurs through the natural consumption of contaminated feed. In this study, we investigated the use of feed dust collected from experimentally inoculated feed as a novel diagnostic sample type for ASFV detection. Moist swabs were used to collect dust from creep feeders after natural consumption of feed inoculated with 3.1–5.4 log₁₀ TCID₅₀/g ASFV Georgia 2007 in the presence and absence of antimicrobial feed additives. Results validate the potential use of feed dust swabs as a novel diagnostic surveillance tool for detection and quantification of viral nucleic acid and infectious virus titre in ASFV-contaminated feed.     

New perspectives for evaluating relative risks of African swine fever virus contamination in global feed ingredient supply chains

There are no published reports indicating that the African swine fever virus (ASFV) has been detected in feed ingredients or complete feed. This is primarily because there are only a few laboratories in the world that have the biosecurity and analytical capabilities of detecting ASFV in feed. Several in vitro studies have been conducted to evaluate ASFV concentration, viability and inactivation when ASFV was added to various feed ingredients and complete feed. These inoculation studies have shown that some feed matrices support virus survival longer than others and the reasons for this are unknown. Current analytical methodologies have significant limitations in sensitivity, repeatability, ability to detect viable virus particles and association with infectivity. As a result, interpretation of findings using various measures may lead to misleading conclusions. Because of analytical and technical challenges, as well as the lack of ASFV contamination data in feed supply chains, quantitative risk assessments have not been conducted. A few qualitative risk assessments have been conducted, but they have not considered differences in potential scenarios for ASFV contamination between various types of feed ingredient supply chains. Therefore, the purpose of this review is to provide a more holistic understanding of the relative potential risks of ASFV contamination in various global feed ingredient supply chains and provide recommendations for addressing the challenges identified.     

The risk and mitigation of foot-and-mouth disease virus infection of pigs through consumption of contaminated feed

Transboundary movement of animal feed and feed ingredients has been identified as a route for pathogen incursions. While imports of animals and animal-derived products are highly regulated for the purpose of infectious disease prevention, there has been less consideration of the viability of infectious agents in inanimate products, such as feed. This study investigated the ability of foot-and-mouth disease virus (FMDV) to remain infectious as a contaminant of commercial whole pig feed and select pig feed ingredients, and to establish the minimum infectious dose (MID_F) required to cause foot-and-mouth disease (FMD) in pigs that consumed contaminated feed. FMDV viability in vitro varied depending on virus strain, feed product, and storage temperature, with increased duration of infectivity in soybean meal compared to pelleted whole feed. Specifically, both strains of FMDV evaluated remained viable through to the end of the 37 day observation period in experimentally contaminated soybean meal stored at 4 or 20°C . The MID_F for pigs consuming contaminated feed varied across virus strains and exposure duration in the range of 10^6.2 to 10⁷ TCID₅₀. The ability of FMDV to cause infection in exposed pigs was mitigated by pre-treatment of feed with two commercially available feed additives, based on either formaldehyde (SalCURB®) or lactic acid (Guardian™). Our findings demonstrate that FMDV may remain infectious in pig feed ingredients for durations compatible with transoceanic transport. Although the observed MID_F was relatively high, variations in feeding conditions and biophysical characteristics of different virus strains may alter the probability of infection. These findings may be used to parameterize modelling of the risk of FMDV incursions and to regulate feed importation to minimize the risk of inadvertent importation.     

Biological characterization of African swine fever virus genotype II strains from north‐eastern Estonia in European wild boar

Due to its impact on animal health and pig industry, African swine fever (ASF) is regarded as one of the most important viral diseases of pigs. Following the ongoing epidemic in the Transcaucasian countries and the Russian Federation, African swine fever virus was introduced into the Estonian wild boar population in 2014. Epidemiological investigations suggested two different introductions into the southern and the north‐eastern part of Estonia. Interestingly, outbreak characteristics varied considerably between the affected regions. While high mortality and mainly virus‐positive animals were observed in the southern region, mortality was low in the north‐eastern area. In the latter, clinically healthy, antibody‐positive animals were found in the hunting bag and detection of virus was rare. Two hypotheses could explain the different behaviour in the north‐east: (i) the frequency of antibody detections combined with the low mortality is the tail of an older, so far undetected epidemic wave coming from the east, or (ii) the virus in this region is attenuated and leads to a less severe clinical outcome. To explore the possibility of virus attenuation, a re‐isolated ASFV strain from the north‐eastern Ida‐Viru region was biologically characterized in European wild boar. Oronasal inoculation led to an acute and severe disease course in all animals with typical pathomorphological lesions. However, one animal recovered completely and was subsequently commingled with three sentinels of the same age class to assess disease transmission. By the end of the trial at 96 days post‐initial inoculation, all animals were completely healthy and neither virus nor viral genomes were detected in the sentinels or the survivor. The survivor, however, showed high antibody levels. In conclusion, the ASFV strain from north‐eastern Estonia was still highly virulent but nevertheless, one animal recovered completely. Under the experimental conditions, no transmission occurred from the survivor to susceptible sentinel pigs.     

Infection of pigs with African swine fever virus via ingestion of stable flies (Stomoxys calcitrans)

Within Eastern Europe, African swine fever virus (ASFV ) has unexpectedly spread to farms with high biosecurity. In an attempt to explain this process, pigs were allowed to ingest flies that had fed on ASFV ‐spiked blood, which had a realistic titre for an infected pig. Some of the pigs became infected with the virus. Thus, ingestion of blood‐sucking flies, having fed on ASFV ‐infected wild boar before entering stables, represents a potential route for disease transmission.     

Quantitative Risk Assessment for the Introduction of African Swine Fever Virus into the European Union by Legal Import of Live Pigs

The recent incursion and spread of African swine fever virus (ASFV) in the Russian Federation and Caucasus region, close to European Union (EU) borders, have increased the concerns regarding the probability of ASFV introduction into the EU. There are many potential routes of ASFV entry into EU, but here we specifically aimed to assess the probability of ASFV introduction by legal trade of pigs, which historically has been one of the most important ways of exotic diseases introduction into the EU. A stochastic model was used to estimate the monthly probability of ASFV introduction for each country of the EU. Results of this model suggest an annual probability for ASFV introduction in the whole EU by this way of 5.22*10⁻³, which approximately corresponds with one outbreak in 192 years. The risk of ASFV introduction via live pigs was highest in Poland (69%), particularly during the months of November and December. As expected, Russian Federation is the country that most contributes to this risk, representing 68% of the overall annual risk. Methods and results presented here may be useful for informing risk‐based surveillance and control programmes and, ultimately, for prevention and control of potential ASFV incursions into the EU.     

Feed or feed transport as a potential route for a porcine epidemic diarrhoea outbreak in a 10,000-sow breeding herd in Mexico

Porcine epidemic diarrhoea virus (PEDV) infects pigs of all ages causing vomiting and diarrhoea. PEDV is transmitted via the oral–faecal route, and a very low dose is enough to infect susceptible pigs, resulting in significant production losses. This short communication aims to describe the introduction of PEDV into a 10,000-sow farrow-to-wean farm located in northwest Mexico. Following the onset of clinical signs, an outbreak investigation was conducted to determine the most probable route of introduction. Based on data collected from interviews, construction of a timeline of events, and the detection of PEDV RNA in feed samples and samples collected from various surfaces of feed transport vehicles, it was concluded that the most probable route for PEDV incursion into this breeding herd was contaminated feed or a contaminated feed transport vehicle. This paper describes how feed or feed transport could serve as potential routes of PEDV infection to a farm and highlights the importance of establishing biosecurity programs to mitigate these risks.     

Selected Swine Viral Pathogens in Indoor Pigs in Spain. Seroprevalence and Farm‐Level Characteristics

A serosurvey on porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), Aujeszky’s disease virus gE protein (ADV gE), porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2) was carried out in Spanish pig herds. The serosurvey consisted of two studies. First, a retrospective study assessed the proportion of seropositive boar, sow and fattening pig herds and their seroprevalences to PRRSV, SIV, ADV gE and PPV from 2003 to 2005 and to PCV2 from 2000 to 2005. Such information was obtained from routine serologic analyses from two veterinary diagnostic laboratory services. Second, a cross‐sectional study in sow and fattening pig herds from 44 farms (without vaccination interferences on serologic analyses) was performed to provide information on seroprevalences and co‐seropositivity to PRRSV, SIV, ADV gE and PCV2 (PPV was excluded because of widespread vaccination) and to elucidate their relationships with farm characteristics, management and productive parameters. Similar seroprevalences were observed in both studies, although some variations were obtained, probably because of vaccination schedules, number of tested sera, sampling age and regional variations. Percentage of PRRSV and SIV seropositive herds was over 85% for sows, around 80% for fatteners and around 50% for boar studs. The proportion of ADV gE seropositive sow herds decreased from 41% to 30% between 2003 and 2005, whereas such decrease was from 41% to 33% in fattening pig herds and from 13% to 4% in boar studs PCV2 antibodies were widespread as well as those against PPV; in the latter case, if antibodies were elicited by infection and/or vaccination was not assessed. Concurrent presence of PCV2, PRRSV and SIV antibodies was found in 89% and 66% sow and fattening herds, respectively. No statistical associations were obtained between seroprevalences or co‐seropositivity and farm characteristics, management or productive parameters.     

Rethinking the uncertainty of African swine fever virus contamination in feed ingredients and risk of introduction into the United States

Economically relevant pathogens, such as African swine fever virus (ASFV), have been shown to survive when experimentally inoculated in some feed ingredients under the environmental conditions in transoceanic transport models. However, these models did not characterize the likelihood of virus survival under various time and temperature processes that feed ingredients undergo before they are added to swine diets. Here, we developed a quantitative risk assessment model to estimate the probability that one or more corn or soybean meal ocean vessels (25,000 tonnes) contaminated with ASFV would be imported into the United States annually. This final probability estimate was conditionally based on five likelihoods: the probability of initial ASFV contamination (p0), ASFV inactivation during processing (p1) and transport (p2), recontamination (pR), and ASFV inactivation while awaiting customs clearance at United States entry (p3). The probability of ASFV inactivation was modelled using corn and soybean (extruded or solvent extracted) processing conditions (times and temperatures), D-values (time to reduce 90% or 1-log) estimated from studies of ASFV thermal inactivation in pork serum (p1), and survival in feed ingredients during transoceanic transport (p2 and p3). ‘What-if’ scenarios using deterministic values for p0 and pR (1%, 10%, 25%, 50%, 75%, and 100%) were used to explore their impact on risk. The model estimated complete inactivation of ASFV after extrusion or solvent extraction processes regardless of the initial ASFV contamination probability assumed. The value of recontamination (ranging from 1% to 75%) was highly influential on the risk of one ASFV-contaminated soybean meal vessel entering the United States. Median risk estimates ranged from 0.064% [0.006%–0.60%; 95% probability interval (PI)], assuming a pR of 1.0%, up to 4.67% (0.45%–36.50% 95% PI) assuming a pR of 75.0%. This means that at least one vessel with ASFV-contaminated soybean meal would be imported once every 1563–21 years, respectively. When all raw corn was assumed to be contaminated (p0 = 100%), and no recontamination was assumed to occur (pR = 0%), the median probability of one vessel with ASFV-contaminated corn entering the United States was 2.02% (0.28%–9.43% 95% PI) or once every 50 years. Values of recontamination between 1% and 75% did not substantially change the risk of corn. Days of transport, virus survival during transport (D-value), and number of vessels shipped were the parameters most influential for increased likelihood of a vessel with ASFV-contaminated soybean meal or corn entering the United States. The model helped to identify knowledge gaps that are most influential on output values and serves as a framework that could be updated and parameterized as new scientific information becomes available. We propose that the quantitative risk assessment model developed in this study can be used as a framework for estimating the risk of ASFV entry into the United States and other ASFV-free countries through other types of imported feed ingredients that may potentially become contaminated. Ultimately, this model can be used to develop risk mitigation strategies and critical control points for inactivating ASFV during feed ingredient processing, storage, and transport, and contribute to the design and implementation of biosecurity measures to prevent the introduction of ASFV into the United States and other ASFV-free countries.     

No evidence for long‐term carrier status of pigs after African swine fever virus infection

This study targeted the assessment of a potential African swine fever virus (ASFV) carrier state of 30 pigs in total which were allowed to recover from infection with ASFV “Netherlands'86” prior exposure to six healthy sentinel pigs for more than 2 months. Throughout the whole trial, blood and swab samples were subjected to routine virological and serological investigations. At the end of the trial, necropsy of all animals was performed and viral persistence and distribution were assessed. Upon infection, a wide range of clinical and pathomorphological signs were observed. After an initial acute phase in all experimentally inoculated pigs, 66.6% recovered completely and seroconverted. However, viral genome was detectable in blood samples for up to 91 days. Lethal outcomes were observed in 33.3% of the pigs with both acute and prolonged courses. No ASFV transmission occurred over the whole in‐contact phase from survivors to sentinels. Similarly, infectious ASFV was not detected in any of the tissue samples from ASFV convalescent and in‐contact pigs. These findings indicate that the suggested role of ASFV survivors is overestimated and has to be reconsidered thoroughly for future risk assessments.     

African swine fever virus survival in buried wild boar carcasses

Since the first introduction of African swine fever (ASF) into the European wild boar population in 1957, the question of virus survival in carcasses of animals that succumbed to the disease has been discussed. The causative African swine fever virus (ASFV) is known to be very stable in the environment. Thus, carcasses of infected wild boar could play a major role as ASFV reservoir and thereby help to locally maintain and spread the disease in wild boar populations. To minimize this risk, removal of wild boar carcasses in ASF affected areas is regarded to be crucial for effective disease control. If removal is not feasible, carcasses are usually disposed by burial on the spot to avoid direct contact of wild boar to the infection source. In this study, carcasses of ASFV infected wild boar buried in Lithuania at different time points and locations have been excavated and retested for the presence of infectious ASFV by in vitro assays and for viral genome by qPCR. Soil samples potentially contaminated by body fluids have been additionally tested for viral genome. In seventeen out of twenty burial sites, samples of excavated carcasses were positive for ASFV genome. However, in none of the carcass samples ASFV could be isolated. On seven sites soil samples contained ASF viral DNA. These results unexpectedly negate the long‐term persistence of infectious ASFV in wild boar carcasses independent from the burial time. In this context, sensitivity of ASFV isolation from carcass samples versus susceptibility of animals and doses needed for oral inoculation has to be further investigated. Furthermore, research is required to consider alternative ASF infection sources and drivers in the infection cycle among wild boar.     

Survival of African Swine Fever Virus in Excretions from Pigs Experimentally Infected with the Georgia 2007/1 Isolate

African swine fever virus (ASFV) causes a lethal haemorrhagic disease of swine which can be transmitted through direct contact with infected animals and their excretions or indirect contact with contaminated fomites. The shedding of ASFV by infected pigs and the stability of ASFV in the environment will determine the extent of environmental contamination. The recent outbreaks of ASF in Europe make it essential to develop disease transmission models in order to design effective control strategies to prevent further spread of ASF. In this study, we assessed the shedding and stability of ASFV in faeces, urine and oral fluid from pigs infected with the Georgia 2007/1 ASFV isolate. The half‐life of infectious ASFV in faeces was found to range from 0.65 days when stored at 4°C to 0.29 days when stored at 37°C, while in urine it was found to range from 2.19 days (4°C) to 0.41 days (37°C). Based on these half‐lives and the estimated dose required for infection, faeces and urine would be estimated to remain infectious for 8.48 and 15.33 days at 4°C and 3.71 and 2.88 days at 37°C, respectively. The half‐life of ASFV DNA was 8 to 9 days in faeces and 2 to 3 days in oral fluid at all temperatures. In urine, the half‐life of ASFV DNA was found to be 32.54 days at 4°C decreasing to 19.48 days at 37°C. These results indicate that ASFV in excretions may be an important route of ASFV transmission.     

Experimental Infection of Domestic Pigs with African Swine Fever Virus Lithuania 2014 Genotype II Field Isolate

An experimental infection was conducted to evaluate horizontal transmission, clinical, virological and humoral response induced in domestic pigs infected with African swine fever (ASF) genotype II virus circulating in 2014 into the European Union (EU). Ten naive pigs were placed in contact with eight pigs experimentally inoculated with the Lithuanian LT14/1490 ASF virus (ASFV) responsible for the first ASF case detected in wild boar in Lithuania in January 2014. Clinical examination and rectal temperature were recorded each day. Blood sampling from every animal was carried out twice weekly. Blood samples were examined for presence of ASF virus‐specific antibodies and for determining the ASFV viral load. From the obtained results, it was concluded that the Lithuanian ASFV induced an acute disease which resulted in 94, 5% mortality. The disease was easily detected by real‐time PCR prior to the onset of clinical signs and 33% of the animals seroconverted. All findings were in accordance with observations previously made in domestic pigs and wild boar when infected with ASF genotype II viruses characterized by a high virulence. One in‐contact pig remained asymptomatic and survived the infection. The role of such animals in virus transmission would need further investigation.     

Inferring within‐herd transmission parameters for African swine fever virus using mortality data from outbreaks in the Russian Federation

Mortality data are routinely collected for many livestock and poultry species, and they are often used for epidemiological purposes, including estimating transmission parameters. In this study, we infer transmission rates for African swine fever virus (ASFV ), an important transboundary disease of swine, using mortality data collected from nine pig herds in the Russian Federation with confirmed outbreaks of ASFV . Parameters in a stochastic model for the transmission of ASFV within a herd were estimated using approximate Bayesian computation. Estimates for the basic reproduction number varied amongst herds, ranging from 4.4 to 17.3. This was primarily a consequence of differences in transmission rate (range: 0.7–2.2), but also differences in the mean infectious period (range: 4.5–8.3 days). We also found differences amongst herds in the mean latent period (range: 5.8–9.7 days). Furthermore, our results suggest that ASFV could be circulating in a herd for several weeks before a substantial increase in mortality is observed in a herd, limiting the usefulness of mortality data as a means of early detection of an outbreak. However, our results also show that mortality data are a potential source of data from which to infer transmission parameters, at least for diseases which cause high mortality.     

Investigation into the Epidemiology of African Swine Fever Virus at the Wildlife – Domestic Interface of the Gorongosa National Park,Central Mozambique

An epidemiological study of African swine fever (ASF) was conducted between March 2006 and September 2007 in a rural area adjacent to the Gorongosa National park (GNP) located in the Central Mozambique. Domestic pigs and warthogs were sampled to determine the prevalence of antibodies against ASF virus and the salivary antigens of Ornithodoros spp. ticks, while ticks collected from pig pens were tested for the presence of ASFV. In addition, 310 framers were interviewed to gain a better understanding of the pig value chain and potential practices that could impact on the spread of the virus. The sero‐prevalence to ASFV was 12.6% on farms and 9.1% in pigs, while it reached 75% in warthogs. Approximately 33% of pigs and 78% of warthogs showed antibodies against salivary antigens of ticks. The differences in sero‐prevalence between farms close to the GNP, where there is greater chance for the sylvatic cycle to cause outbreaks, and farms located in the rest of the district, where pig to pig transmission is more likely to occur, were marginally significant. Ornithodoros spp. ticks were found in only 2 of 20 pig pens outside the GNP, and both pens had ticks testing positive for ASFV DNA. Interviews carried out among farmers indicated that biosecurity measures were mostly absent. Herd sizes were small with pigs kept in a free‐ranging husbandry system (65%). Only 1.6% of farmers slaughtered on their premises, but 51% acknowledged allowing visitors into their farms to purchase pigs. ASF outbreaks seemed to have a severe economic impact with nearly 36% of farmers ceasing pig farming for at least 1 year after a suspected ASF outbreak. This study provides the first evidence of the existence of a sylvatic cycle in Mozambique and confirms the presence of a permanent source of virus for the domestic pig value chain.     

Stability of classical swine fever virus and pseudorabies virus in animal feed ingredients exposed to transpacific shipping conditions

Classical swine fever virus (CSFV) and pseudorabies virus (PRV) are two of the most significant trade‐limiting pathogens affecting swine worldwide. Both viruses are endemic to China where millions of kilograms of feed ingredients are manufactured and subsequently imported into the United States. Although stability and oral transmission of both viruses through contaminated pork products has been demonstrated as a risk factor for transboundary spread, stability in animal feed ingredients had yet to be investigated. The objective of this study was to determine the survival of CSFV and variant PRV in 12 animal feeds and ingredients exposed to environmental conditions simulating a 37‐day transpacific shipment. Virus was detected by PCR, virus isolation and nursery pig bioassay. CSFV and PRV nucleic acids were stable throughout the 37‐day period in all feed matrices. Infectious CSFV was detected in two ingredients (conventional soybean meal and pork sausage casings) at 37 days post‐contamination, whereas infectious PRV was detected in nine ingredients (conventional and organic soybean meal, lysine, choline, vitamin D, moist cat and dog food, dry dog food and pork sausage casings). This study demonstrates the relative stability of CSFV and PRV in different feed ingredients under shipment conditions and provides evidence that feed ingredients may represent important risk factors for the transboundary spread of these viruses.     

Comparative genomic analysis reveals an ‘open’ pan‐genome of African swine fever virus

The worldwide transmission of African swine fever virus (ASFV) drastically affects the pig industry and global trade. Development of vaccines is hindered by the lack of knowledge of the genomic characteristics of ASFV. In this study, we developed a pipeline for the de novo assembly of ASFV genome without virus isolation and purification. We then used a comparative genomics approach to systematically study 46 genomes of ASFVs to reveal the genomic characteristics. The analysis revealed that ASFV has an ‘open’ pan‐genome based on both protein‐coding genes and intergenic regions. Of the 151–174 genes found in the ASFV strains, only 86 were identified as core genes; the remainder were flexible accessory genes. Notably, 44 of the 86 core genes and 155 of the 324 accessory genes have been functionally annotated according to the known proteins. Interestingly, a dynamic number of taxis‐related genes were identified in the accessory genes, and two potential virulence genes were identified in all ASFV isolates. The ‘open’ pan‐genome of ASFV based on gene and intergenic regions reveals its pronounced natural diversity concerning genomic composition and regulation.     

Monitoring of African Swine Fever in the Wild Boar Population of the Most Recent Endemic Area of Spain

Wild boars are natural hosts for African swine fever (ASF). The ASF virus (ASFV) can persist for long periods in the environment, such as in ticks and contaminated products, which may be sources of infection for wild boar populations. African swine fever was eradicated in domestic pig populations in Spain in 1995, after 35 years of significant effort. To determine whether ASFV can persist in wild boar hosts after it has been eradicated from domestic pigs and to study the role of wild boar in helping ASFV persist in the environment, we checked for the presence of ASFV in wild boars in Doñana National Park, one of the largest natural habitats of wild boar in Spain and one of the last areas where ASF was endemic prior its eradication. Samples from 158 animals collected between 2006 and 2010 were analysed using serological and nucleic acid‐based diagnostic techniques recommended by the World Organization for Animal Health (OIE). None of the samples was found to be positive. These results confirm the absence of disease in wildlife in what was once one of the areas most affected by ASF in Spain, and they suggest that wild boars play a limited role in ASFV persistence. These results confirm that ASFV cannot persist in isolated wild boar populations for long periods of time without the interaction of other factors such as re‐infection by contact with domestic pigs or by feeding on contaminated swill.     

Effect of mixing and feed batch sequencing on the prevalence and distribution of African swine fever virus in swine feed

It is critical to have methods that can detect and mitigate the risk of African swine fever virus (ASFV) in potentially contaminated feed or ingredients bound for the United States. The purpose of this work was to evaluate feed batch sequencing as a mitigation technique for ASFV contamination in a feed mill, and to determine if a feed sampling method could identify ASFV following experimental inoculation. Batches of feed were manufactured in a BSL-3Ag room at Kansas State University's Biosafety Research Institute in Manhattan, Kansas. First, the pilot feed manufacturing system mixed, conveyed, and discharged an ASFV-free diet. Next, a diet was manufactured using the same equipment, but contained feed inoculated with ASFV for final concentration of 5.6 × 10⁴ TCID₅₀/g. Then, four subsequent ASFV-free batches of feed were manufactured. After discharging each batch into a collection container, 10 samples were collected in a double ‘X’ pattern. Samples were analysed using a qPCR assay for ASFV p72 gene then the cycle threshold (Ct) and Log₁₀ genomic copy number (CN)/g of feed were determined. The qPCR Ct values (p < .0001) and the Log₁₀ genomic CN/g (p < .0001) content of feed samples were impacted based on the batch of feed. Feed samples obtained after manufacturing the ASFV-contaminated diet contained the greatest amounts of ASFV p72 DNA across all criteria (p < .05). Quantity of ASFV p72 DNA decreased sequentially as additional batches of feed were manufactured, but was still detectable after batch sequence 4. This subsampling method was able to identify ASFV genetic material in feed samples using p72 qPCR. In summary, sequencing batches of feed decreases concentration of ASFV contamination in feed, but does not eliminate it. Bulk ingredients can be accurately evaluated for ASFV contamination by collecting 10 subsamples using the sampling method described herein. Future research is needed to evaluate if different mitigation techniques can reduce ASFV feed contamination.