African swine fever virus is currently considered the most significant threat to worldwide pork production. Not only does ASFV cause severe disease and high case fatality in infected pigs of all age groups, but also detection of the virus in a single pig can shut down export markets and substantially shift the global trade of pork.

Since the first reported case of ASFV in China in August 2018, the virus has spread rapidly to more than 10 Asian countries, including Vietnam, North Korea, Cambodia, South Korea and the Philippines.

Most recently, in May 2020, India reported its first confirmed outbreak of ASF (NHF 2020) and listed the initial virus introduction as occurring in January 2020 (Lundeen 2020).

Concurrent to the spread of ASFV through Asia over the last two years, the virus was also being detected for the first time in several European countries, such as Belgium, Slovakia, Serbia and Greece.

Most recently, in September 2020, Germany reported its first case of ASFV detected in a dead wild boar located in close proximity to the border of Poland (Durisin, Parkin et al. 2020).

Within days of the announcement of ASF being detected in Germany, China and several other Asian countries, including Taiwan and Japan, banned pork imports and live pigs from Germany (Welshans 2020).

The discovery of ASFV and the resulting bans on pork from
Germany, the largest swine producer in the European Union, will cause substantial shifts in global pork markets. The impact of ASF on pork exports and the importance of preventing entry of the virus into U.S. swine herds cannot be overstated.

Keeping ASFV out
ASFV is a unique virus that presents several distinct challenges to disease control and eradication. First, ASFV is the only member of the virus family Asfarviridae, which precludes the correlation of ASF pathogenesis and protection to known pathologic or immunologic mechanisms of closely related viruses.

ASFV has a genome of 170-190 kilobase pairs encoding for 151-167 proteins (Galindo and Alonso 2017). Compared to influenza virus A, which has a genome of approximately 14 kilobase encoding for 10-14 proteins, ASFV is extremely large and complex.

It is the only DNA arthropod-borne virus (all other arthropod-borne viruses are RNA), and the virus is stable in the environment due to resistance of pH and temperature extremes (Niederwerder and Rowland 2017).

With no commercially available vaccine for the prevention of infection nor treatment available to reduce disease severity in infected pigs, the overwhelming objective of negative countries is to effectively prevent ASFV introduction through the maintenance of biosecurity.

As a relatively new area of concern in the biosecurity realm, feed biosecurity has become an important and widely recognized target of biosecurity important for the prevention of porcine viral disease entry onto farms.

First recognized after the introduction of porcine epidemic diarrhea virus into the U.S. in 2013 (Niederwerder and Hesse 2018), imported feed ingredients and the resulting feed are now acknowledged as potential sources of swine viruses and should be considered as a part of all farm biosecurity plans.

Considering the risk of ASF, our previous work has demonstrated that the virus is broadly stable in commonly imported feed ingredients subjected to a transoceanic shipment model.

Specifically, infectious ASFV was detected in nine of the 12 ingredients we tested (conventional soybean meal, organic soybean meal, soy oil cake, choline, moist cat food, moist dog food, dry dog food, pork sausage casings and complete feed) in an experimental lab setting after 30 days of fluctuating temperature and humidity environmental conditions (Dee, Bauermann et al. 2018).

Over the course of the transoceanic shipment period, the time required for ASFV to decay by approximately half its original concentration was between 9.6 and 14.2 days, with an average half-life of 12.2 days across all ingredients tested (Stoian, Zimmerman et al. 2019).

Further, we confirmed that ASF is transmissible through the natural consumption of contaminated plant-based feed, with a minimum infectious dose of 104 TCID50, and an increased likelihood of infection when a contaminated batch of feed is consumed over time (Niederwerder, Stoian et al. 2019).

As thousands of metric tons of feed ingredients are imported each year to the U.S. from countries with active outbreaks of ASF (Patterson, Niederwerder et al. 2020), it is critically important that mitigation strategies are identified to reduce the risk of ASFV entry through this route.

After confirming the stability and transmission of ASFV in feed, the purpose of our latest work (Niederwerder, Dee et al. 2020) summarized herein was to identify mitigation tools which may be utilized to reduce the risk of ASFV introduction and transmission through feed.

Specifically, we sought to determine the efficacy of two classes of feed additives on inactivating or reducing infectivity of ASFV.

Feed additive classes included in the study were medium-chain fatty acids (MCFA, 1:1:1 ratio of C6, C8 and C10) and aqueous formadehyde (Sal CURB, a Kemin product) liquid antimicrobial products, which were selected due to previous experimental work demonstrating efficacy on other viruses endemic to U.S. swine.

MCFAs, formaldehyde
Mechanistically, these antimicrobial products are considered to inactivate viruses in two different ways. First, MCFAs are believed to reduce virus infectivity by disruption of the viral envelope, leading to deconstruction of the virion and an inability to bind to the host cell for infection (Thormar, Isaacs et al. 1987).

Second, formaldehyde is believed to reduce virus infectivity through alkylation and cross-linking of viral nucleic acids and proteins (Sabbaghi, Miri et al. 2019).

Efficacy of each feed additive against ASFV was investigated through a twofold approach, including objectives to:

In the first objective, each liquid product was added at various inclusion rates (from 0.03% to 2.0%) to a standard concentration of ASFV BA71V cell culture-adapted virus strain.

Each additive or virus mixture was incubated for up to 30 minutes prior to creating serial dilutions for testing the concentration of virus remaining after exposure to the liquid additive.

The quantity of ASFV remaining after exposure to either MCFAs or formaldehyde-based feed additives was compared to negative (feed additive lacking ASFV) and positive (ASFV lacking feed additives) control samples.

Results of the first objective demonstrated a dose-dependent reduction in ASF virus titer after exposure to MCFAs or formaldehyde-based feed additives.

Both liquid antimicrobial products were effective at reducing ASFV infectivity in cell culture, with inclusion rates defined for MCFAs (0.7%) and formaldehyde-based feed additives (0.35%) that were required to reduce the ASFV quantity below the detection threshold in cell culture.

Transoceanic model
In the latter objective, we tested the efficacy of MCFAs and formaldehyde-based feed additives when mixed into nine different feed ingredients prior to the transoceanic model (Day 0, in the simulated country of origin) and at the end of the transoceanic model (Day 28, in the simulated country of arrival).

The nine feed ingredients were selected due to their known ability to support ASFV for at least 30 days in the transoceanic model (Dee, Bauermann et al. 2018) and included conventional soybean meal, organic soybean meal, soy oil cake, choline, moist dog food, moist cat food, dry dog food, pork sausage casings and complete feed in meal form.

Five grams of each feed ingredient were inoculated with ASFV Georgia 2007 and tested in duplicate after being treated with the feed additives at each of the two time points. Inclusion rates for MCFAs (1.0%) and formaldehyde-based feed additives (0.33%) were selected based on previous work on endemic swine viruses as described above.

All samples were placed in an environmental chamber for exposure to temperature and humidity conditions simulating a real-world 30-day shipment period from Eastern Europe.

Treated feed ingredients were compared with negative (ingredients lacking ASFV inoculation) and positive (ASFV inoculated ingredients lacking feed additives) control samples.

Feed ingredient samples were processed at four time points during the 30-day transoceanic shipment model by adding 15 milliliters of sterile phosphate-buffered saline to feed, and mixing, centrifuging and storing the supernatant fluid for diagnostic testing.

Supernatant samples were tested for the presence of ASFV DNA through polymerase chain reaction and for the presence of infectious ASFV through virus isolation on cell culture or through pig bioassay.

Results of the latter objective demonstrated a reduction of ASFV infectivity in feed ingredients when treated with MCFAs or formaldehyde-based feed additives at Day 0 or Day 28 of the transoceanic shipment model.

The majority of feed ingredient samples (16 of 18) treated with MCFAs and all of the feed ingredient samples (18 of 18) treated with formaldehyde-based feed additives were negative for the presence of infectious virus on both cell culture and in pig bioassay.

Although most samples proved to be negative for infectious ASFV in cell culture or pig bioassay after feed additive treatment, all feed ingredient samples maintained the presence of detectable ASFV DNA (positive PCR test), highlighting the important differences between these diagnostic assays.

Under the conditions of these studies, both MCFAs and formaldehyde-based feed additives demonstrated efficacy in a dose-dependent manner for reducing ASFV infectivity and show promise as potential tools in our toolbox of mitigation strategies for reducing the risk of ASFV introduction and transmission through feed.

This article is based on the published work found at Niederwerder MC, Dee S, Diel DG, et al. Mitigating the risk of African swine fever virus in feed with anti-viral chemical additives. Transbound Emerg Dis. 2020;00:1-10. doi.org/10.1111/tbed.13699.

Niederwerder is an assistant professor in the College of Veterinary Medicine at Kansas State University.

References

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