Swine enteric coronaviruses, including porcine epidemic diarrhea virus and porcine deltacoronavirus, are considered the last major transboundary swine diseases introduced into the U.S. pig population in 2013 and 2014, respectively (Niederwerder and Hesse, 2018).
The causative agents of both diseases are single-stranded enveloped RNA viruses in the family Coronaviridae. In contrast to the currently circulating novel coronavirus (COVID-19) in humans, these swine coronaviruses are major causes of gastrointestinal disease in pigs and are not a major cause of clinical respiratory signs.
Within eight weeks after the first case of PEDV was detected in North America, the virus had spread to most of the major swine-producing regions in the U.S. (Niederwerder and Hesse, 2018). Within one year after the virus was introduced, PEDV was responsible for the loss of 10% of the U.S. swine crop, killing over 7 million pigs.
Death due to this disease is devastating, as the majority of severely affected pigs and mortalities are neonates within the first few days of life. Almost seven years after introduction of PEDV, this virus continues to cause endemic disease in U.S. swine (Swine Health Information Center, 2020), and investigations continue to seek improvement and refinement of disease control protocols.
After the introduction of PEDV, several epidemiological investigations into the genetic sequence and rapid spread of the virus into new states and farms revealed the potential source of virus as contaminated feed and feed ingredients.
First, the genetic sequence of PEDV introduced into the U.S. was over 99% similar to the strain which was circulating in China at the time (Huang, Dickerman et al., 2013). Second, research revealed that PEDV was capable of surviving in several feed ingredients exposed to temperature and humidity conditions which simulated real-world transpacific shipment based on historical meteorological data (Dee, Neill et al., 2016).
Third, experimental research revealed that PEDV could be transmitted through the natural consumption of contaminated feed, and that the minimum infectious dose was low (Dee, Clement et al., 2014; Schumacher, Woodworth et al., 2016). Fourth, PEDV genome was detected on polymer chain reaction testing of feed samples that had been implicated as potential sources of the virus onto new farms in Ohio and Canada (Pasick, Berhane et al., 2014; Bowman, Krogwold et al., 2015).
Taken together, the risk of feed ingredients as a factor for disease introduction onto swine farms was recognized due to the North American experience with PEDV.
When considering feed ingredients as a potential pathogen source, there are several factors affecting risk. First, the country of origin for feed ingredients plays a major role; and it is important to know what swine diseases are currently circulating, including outbreaks in specific regions or endemic diseases of widespread prevalence.
For example, sourcing ingredients within the U.S. poses significantly fewer risks for foreign animal diseases but does not eliminate the possibility for feed to serve as a vector for endemic diseases already present in the country.
Second, the environmental stability of the virus and the stability of the virus in feed ingredients should be considered. Specifically, experimental research has identified high-risk ingredients which support stability of individual viruses.
Of the 14 viruses which have been tested in transoceanic shipment models to date (Stoian, Petrovan et al., 2020), feed ingredients which support broad and diverse virus stability have also been revealed. These ingredients are important to consider for those pathogens not yet tested in the model as well as those pathogens yet to emerge or be identified.
Third, the agricultural practices or manufacturing practices which are required to produce the ingredient should be considered. For example, the practice of drying grains in China on roadways that are shared by trucks transporting swine increases risk. In contrast, products manufactured in high-biosecurity facilities with lower likelihood of environmental exposure and contamination pose fewer risks.
ASFV, CFSV and PRV
Once feed and feed ingredients were identified as a possible novel route by which transboundary viruses were being spread, the focus of the work in our laboratory has been to characterize feed as a risk for three of the most significant FADs to the U.S. swine industry, including African swine fever virus, classical swine fever virus and pseudorabies virus.
ASFV is an enveloped double-stranded DNA virus and the only member of the family Asfarviridae. CSFV is an enveloped positive-sense RNA virus in the family Flaviviridae, and PRV is an enveloped double-stranded DNA virus in the family Herpesviridae.
Recent changes in the geographic distribution or strain virulence have heightened global concerns for these transboundary viruses. On the SHIC Swine Disease Matrix (SHIC, 2018), ASFV, CSFV and PRV rank Nos. 2, 3 and 4, respectively, with regards to three ranking criteria, including 1) likelihood of entry; 2) impact on production post-entry; and 3) impact on trade and agricultural markets.
Housed on the Kansas State University campus, the Biosecurity Research Institute provides Biosafety Level 3 laboratory and Biosafety Level 3 Agriculture research facilities, affording the unique opportunity to safely and securely perform research directly on these priority pathogens without the need for surrogate viruses.
High-risk feed ingredients
Research on ASFV, CSFV and PRV risk in feed has focused on three priority objectives. The initial goal was to define the high-risk feed ingredients which support ASFV, CSFV and PRV stability during exposure to fluctuating temperature and humidity conditions simulating transoceanic shipment. Twelve feeds or feed ingredients were inoculated with virus, subjected to transoceanic shipping conditions, and tested for the presence of infectious virus at the conclusion of the simulated shipment model.
Across a 30-day transatlantic shipment model, ASFV Georgia 2007 was broadly stable across feed ingredients, with infectious virus being detected in 75% (9/12) of the feed ingredients tested, including conventional soybean meal, organic soybean meal, soy oilcake, choline, moist cat food, moist dog food, dry dog food, pork sausage casings and complete feed (Dee, Bauermann et al., 2018).
Similarly across a 37-day transpacific shipment model, PRV HeN1 Chinese variant was also broadly stable across a wide range of feed ingredients, with viable virus detected in 75% (9/12) of the tested feed ingredients, including conventional soybean meal, organic soybean meal, lysine, choline, vitamin D, moist cat food, moist dog food, dry dog food and pork sausage casings (Stoian, Petrovan et al., 2020).
Lastly, when CSFV Brescia was inoculated into feed ingredients and subjected to the 37-day transpacific shipment model, infectious virus was detected in 17% (2/12) of the tested feed ingredients at the conclusion of the shipment period, including conventional soybean meal and pork sausage casings (Stoian, Petrovan et al., 2020).
Overall, ASFV, CSFV and PRV all survived in feed ingredients in models of transoceanic shipment, with ASFV and PRV having broad stability across a wide range of ingredients. Importantly, all three viruses survived in two ingredients identified as high risk for these FADs, including conventional soybean meal and pork sausage casings.
Minimum infectious dose
The second objective of the three-part approach was to determine the minimum infectious dose and transmissibility of ASFV when consumed naturally through normal feeding and drinking behaviors (Niederwerder, Stoian et al., 2019).
To investigate this objective, 84 nursery pigs were divided into a liquid media or complete feed group; groups were allowed to naturally consume 100 ml of ASFV-contaminated liquid or 100 grams of ASFV-contaminated feed, respectively. Individual pigs consumed ASFV Georgia 2007 at doses between 100 and 108 50% tissue culture infectious dose (TCID50).
Overall, results of the study confirmed ASFV is transmissible through the natural consumption of contaminated plant-based feed and through the natural drinking of contaminated liquid. Higher doses of the virus were required for infection through plant-based feed when compared to liquid, which we believe is due to increased virus contact with the tonsils, an early replication site for the virus when consumed in a liquid matrix.
Specifically, the minimum infectious dose of ASFV in liquid was 100 TCID50, whereas 104 TCID50 was the minimum infectious dose of ASFV in plant-based feed. Modeling repeated exposures over time (i.e., consumption of a contaminated batch of feed or a contaminated water source) revealed an increased likelihood of infection even at low doses.
Taken together, ASFV can be transmitted orally via natural consumption of contaminated plant-based feed, with infection probability dependent on dose and number of exposures (Niederwerder, Stoian et al., 2019).
Mitigating the risk
The third objective of the three-part approach is ongoing and is focused on assessing tools for mitigating the risk of ASFV transmission in feed. As the first step in this objective, our initial goal was to define the half-life of ASFV Georgia 2007 in the nine feeds or feed ingredients which promoted viral stability in transoceanic shipment conditions (Stoian, Zimmerman et al., 2019).
Half-life is defined as the time necessary for the quantity of virus to be reduced by half of its initial concentration. To determine ASFV half-life in feed ingredients, the transoceanic model was replicated, and virus titers quantified at one, eight, 17 and 30 days post-contamination, resulting in a total of 144 titrations across nine feed matrices.
Using the virus quantifications, we characterized viral decay over time and calculated robust half-life estimates, which included standard errors and confidence intervals. ASFV half-life estimates across all feed ingredients tested in the transoceanic shipment model were between 9.6 and 14.2 days, with an average half-life of 12.2 days.
Of concern was the discovery that all nine feed matrices increased ASFV stability when compared to the virus stability in laboratory media, where the shortest half-life was calculated at 8.3 days (Stoian, Zimmerman et al., 2019). Variability in half-life estimates between feed matrices is likely associated with protein, fat or moisture content.
Recently, these half-life estimates were used to provide mean holding time calculations for 99.99% degradation of ASFV in high-risk feed ingredients (SHIC, 2020). Thirteen half-lives are required to reduce the virus concentration to 0.01% of its initial quantity. Mean holding times ranged between 125 and 168 days based on calculations for conventional soybean meal, organic soybean meal and choline.
Overall, the feed matrix promotes ASFV stability, and approximately two weeks are required for the virus concentration to decline by half under shipping conditions.
Although there are many other risk factors for introduction of ASFV and other FADs into the U.S. swine herd, feed ingredients are of particular concern due to their intended purpose and distinct access to commercial pigs in high-biosecurity farms.
Considerations on how to reduce the risk of feed and feed ingredients as vehicles for FADs into the U.S. swine population should be a high priority and start with characterizing the necessity, source and risk for each feed ingredient.
Current swine farm biosecurity protocols should be translated to the feed mill environment and include limitations on access of people and vehicles, lines of separation to identify restricted areas, disinfection protocols, quarantine time for employees or visitors after international travel to high-risk areas or exposure to swine, pest control, and employee training on safe feed handling.
It is estimated that introduction of ASFV into the U.S. swine herd would cause economic losses in excess of $10 billion due to production losses and market disruption. Preventing FAD introduction into U.S. livestock is paramount to maintaining the agricultural economy and food supply.
Due to the high cost of introduction, significant resources should be invested toward preventative measures and research into best practices for mitigating the risk of FAD through feed.
Niederwerder is an assistant professor in the College of Veterinary Medicine at Kansas State University.