The risk of viral transmission in feed: What do we know, what do we do?

Abstract The role of animal feed as a vehicle for the transport and transmission of viral diseases was first identified in 2014 during the porcine epidemic diarrhoea virus epidemic in North America. Since the identification of this novel risk factor, scientists have conducted numerous studies to understand its relevance. Over the past few years, the body of scientific evidence supporting the reality of this risk has grown substantially. In addition, numerous papers describing actions and interventions designed to mitigate this risk have been published. Therefore, the purpose of this paper is to review the literature on the risk of feed (what do we know) and the protocols developed to reduce this risk (what do we do) in an effort to develop a comprehensive document to raise awareness, facilitate learning, improve the accuracy of risk assessments and to identify knowledge gaps for future studies.


| Introduction
Effective biosecurity protocols are essential towards protecting the health status of swine farms. In the United States, tremendous resources have been invested to reduce the risk of viral pathogens, such as the entry of porcine reproductive and respiratory syndrome virus into susceptible populations. Protocols including shower in-shower out, transport sanitation, quarantine and testing of incoming genetics and the filtration of incoming air are commonplace throughout the US swine industry, particularly at the level of the sow farm (Silva, Corbellini, Linhares, Baker, & Holtkamp, 2018). In contrast, prior to the May 2013 entry of porcine epidemic diarrhoea virus (PEDV) into the US swine population (Chen et al., 2014) the role of swine feed as a vehicle for pathogen transport and transmission had not been considered, despite the fact that feed is delivered to swine farms on a daily basis in the absence of any biosecurity protocols. Since the identification of this novel risk factor, scientists across North America have conducted numerous studies to understand its relevance. Therefore, the purpose of this paper is to review the literature on the risk of feed (what do we know?) and the protocols that have been developed to reduce this risk (what do we do?), in an effort to develop a comprehensive document to raise awareness, facilitate learning and identify knowledge gaps for future studies.

| PEDV changes the paradigm
Upon its entry to the United States, PEDV spread rapidly throughout the country at an unprecedented rate (Niederwerder & Hesse, 2018).
Following phylogenetic analysis, it was determined that the virus most likely originated from China (Huang et al., 2013). During the initial outbreak, the American Association of Swine Veterinarians, the National Pork Producers Council and the USDA Center for Epidemiology and Animal Health conducted an epidemiological investigation involving porcine epidemic diarrhoea (PED) affected case and control herds. Of the more than 100 variables surveyed during the investigation, seven were significantly associated with acquiring PEDV during the process of feeding animals, including using sow feed that was custom mixed off-farm in the last 90 days prior to the questionnaire, how many meal/mash rations were fed to nursery or finishers in the last 90 days, the total number of different rations fed to finisher pigs in the last 90 days, the contents in terms of supplementation that was in the premix for the most recent finisher diet and what type of grain mix was used for sow or finisher feed in the past 90 days (AASV, NPPC & USDA CEAH, 2015).
In 2014, the risk was confirmed when ingestion of contaminated complete feed was proven to be a vehicle for PEDV transmission to naïve pigs . This study involved the detection of viral nucleic acid in feed dust samples from the interior walls of feed bins that had provided feed for the index cases of PED in sows across a subset of farms, followed by a demonstration of virus viability in a pig bioassay model through natural feeding behaviour . Within 3-4 days after consumption, evidence of PEDV infection was noted, including clinical signs of PED (vomiting and diarrhoea), detection of PEDV RNA in rectal swabs and PED-consistent lesions in the gastrointestinal tract. This publication resulted in a series of laboratory experiments to validate the results and expand upon the concept of feed and feed ingredients as risk factors for viral transport and transmission. It must be noted that while the original study  used naturally contaminated feed, the majority of these follow up experiments involved purposeful inoculation (spiking) of ingredients.
Following the proof of concept study, the minimum infectious dose of PEDV in feed was determined to be 5.6 × 10 1 TCID 50 /g using the 10-day-old piglet bioassay (Schumacher et al., 2016). In addition, the potential for widespread PEDV contamination of surfaces in an animal food manufacturing facility was evaluated . In this study, a U.S. virulent PEDV isolate was used to inoculate 50 kg of swine feed, which was then mixed, conveyed and discharged into bags using pilot-scale feed manufacturing equipment. Subsequent collection of environmental swabs demonstrated widespread distribution of virus via feed dust, with the presence of PEDV ribonucleic acid (RNA) in 100% of dust samples collected from animal food-contact surfaces and in 89% of dust samples from non-animal food-contact surfaces. Once contamination of the feed mill environment was demonstrated, the question of whether viral survival would differ across the various feed ingredients found in a milling environment was investigated (Dee et al., 2015). A subset of feed ingredients used in swine rations were inoculated with PEDV and stored outdoors during the month of January in Minnesota.
Interestingly, viable PEDV was detected by virus isolation or swine bioassay out to 180 days post-inoculation (DPI) in conventional (high protein/low fat) soybean meal, as well as out to 30 DPI in DDGS, meat and bone meal, RBCs, lysine HCL, D/L methionine, choice white grease, choline chloride, and out to 7 DPI in limestone and 14 DPI in threonine. In contrast, viable PEDV was not present in several other ingredients, including corn, various animal protein sources and vitamin/trace mineral mixes (Dee et al., 2015).  (Scott et al., 2016). Surprisingly, the virus did not survive in the absence of a feed matrix, suggesting that survival is dependent upon the presence of the ingredient, not the container (tote) per se.

| Expanding the viral portfolio
Based on these collective data involving PEDV, the transboundary experiment was repeated across 11 other viruses, including Senecavirus The ASFV outbreak in China accelerated the research efforts to better understand the risk of feed, specifically as it pertained to ASFV.
This resulted in the work of Niederwerder and others who documented transmission of ASFV to naïve pigs following natural consumption of purposefully contaminated feed and liquid . This study determined the minimum infectious dose of ASFV in liquid (10 0 TCID 50 ) and in feed (10 4 TCID 50 ) following a one-time exposure. However, further analysis indicated that the more frequent the exposure (3×, 10×, 30×) to ASFV in small volumes of feed or liquid, the higher the probability of infection, even in the presence of lower doses such as 10 2 TCID 50 . Another significant finding was the calculation of  (Stoian et al., 2020). Surprisingly, viable PRV was recovered from 9 ingredients (conventional soybean meal, organic soybean meal, lysine, choline, vitamin D, moist and dry pet food, and pork sausage casings) while in contrast, viable CSFV was only recovered from conventional soybean meal and pork sausage casings.

| PART 2: WHAT DO WE DO?
Since the discovery of PEDV in the United States and the role that

| Strategy 1: Mechanical reduction (flushing and sequencing)
Several experiments have been conducted to assess the efficacy of decontaminating feed and feed manufacturing facilities through the physical process of mixing, using repeated sequencing of clean feed following known contaminated batches or through the use of chemically treated rice hulls Schumacher et al., 2018) In regards to sequencing, results demonstrated that sequenced batches of feed had reduced quantities of PEDV RNA, although sequenced feed without detectible PEDV RNA was still infectious (Schumacher et al., 2018). Therefore, this protocol can reduce but not eliminate the risk of producing infectious PEDV carryover from the first batch of feed. In regards to the use of chemically treated rice hulls, flushes treated with formaldehyde or mediumchain fatty acid (MCFA) blends reduced the quantity of detectible RNA present after mixing a batch of PEDV-positive feed .

| Strategy 2: Heat treatment
Several studies have demonstrated a positive effect of temperature on the inactivation of PEDV in feed Gerber et al., 2014;Trudeau et al., 2016;. Early work on the effect of heat treatment by Trudeau et al indicated that heating swine feed at temperatures over 130°C effectively reduced PEDV survival (Trudeau et al., 2016;. Furthermore, the spray drying process also was effective in inactivating infectious PEDV in plasma protein (Gerber et al., 2014). Finally, in regards to pelleting, conditioning and pelleting temperatures above 54.4°C were effective in reducing the quantity and infectivity of PEDV in swine feed . In contrast, viable virus was present following exposure to lower (37.8°C and 46.1°C) conditioning temperatures.

| Strategy 3: Chemical mitigation
Extensive studies have been conducted to evaluate the effect of chemical mitigation on PEDV-contaminated feed (Cochrane et al., 2019;Dee, Neill, Clement, Christopher-Hennings, & Nelson, 2014;Huss et al., 2017;Trudeau et al., 2016). The initial work re- In contrast, no evidence of infection was observed in pigs fed Sal CURB ® -treated feed (Dee, Neill, et al., 2014). In another study, feed samples were spiked with PEDV and mixed with either organic acid mixtures, sugar or salt and were incubated at room temperature for up to 21 days. All additives tested were effective in reducing the survival of PEDV as compared to non-treated controls (Trudeau et al., 2016). Recent work by Cochrane and others compared the efficacy of MCFA to other common fat sources to minimize infectivity of feed contaminated with PEDV (Cochrane et al., 2019). Results indicated that feed treated with individual MCFA, 1% MCFA blend or formaldehyde had less detectable viral RNA than other treatments, such as canola oil, coconut oil, palm kernel oil and choice white grease. In addition, PEDV-contaminated feed treated with formaldehyde, 1% MCFA, 0.66% caproic, 0.66% caprylic and 0.66% capric significantly reduced infectivity, in contrast to feed treated with C12 or longer chain fatty acid sources.
In regards to the elimination of PEDV from a contaminated animal feed manufacturing facility, the combined application of a quaternary ammonium-glutaraldehyde blend cleaner, followed by a sodium hypochlorite sanitizing solution, along with a facility heat-up to 60°C for 48 hr was effective at reducing PEDV genomic material, but did not completely eliminate it, demonstrating the residual risk of this virus at the feed mill level following purposeful contamination .

| Strategy 4: Responsible Imports
With the generation of new knowledge on viral half-life in feed, the application of a 'Responsible Imports' approach has been adapted across the US industry (Patterson, Niederwerder, & Dee, 2019).
Responsible Imports, a science-based protocol to safely introduce essential feed ingredients from high-risk countries using extended periods of storage, is based on the following principles: Necessity: is importation of the ingredient an absolute necessity?
Alternatives: can the ingredient be obtained from a country free from foreign animal diseases?

Virus: which virus is causing the concern?
Viral half-life: is there published information on the half-life of the virus in the designated ingredient?
Transport time: what is the projected time for delivery of the ingredient from the source to its destination? Mitigation: are there safe products that can be added to the ingredient to reduce viral load during transport?
Storage period: is there published information on storage time and temperature that will eliminate residual virus from the ingredient prior to use? Therefore, as production companies across the United States develop storage facilities for incoming products, a new way of thinking is taking shape, one that is based on 'feed quarantine' that brings together information across several disciplines including feed science, microbiology and oceanic transport logistics to understand how to minimize risk. This approach is intriguing as it is non-regulatory in nature and does not negatively impact trade.

| CON CLUS I ON S AND NE X T S TEPS
In summary, there is a growing body of scientific evidence suggesting that contaminated feed and feed ingredients purposefully inoculated with viruses may be risk factors for the spread of viral diseases at the domestic and the transboundary levels. This information has stimulated collaborative efforts across North America between livestock and grain commodity groups, governmental agencies, and the veterinary profession in an effort to manage this risk. For example, the Canadian Food Inspection Agency has already implemented a national program using designated secondary control zones to manage the introduction of high-risk feed ingredients, such as grains, However, despite the progress that has been made, significant research gaps still exist regarding the risk of feed. For example, the vast majority of the published papers are based on experimental inoculation and models. Further efforts to reproduce this work using actual modes of transport, that is actual ocean freighters and commercial transport vehicles trucks, are needed. In addition, it is argued that there is a lack of evidence documenting the presence of viral pathogens in actual feed samples around the world. While the current evidence is indeed limited, viable PEDV has been detected in feed bins feeding index cases of PED on sow farms  and ASFV DNA has been detected in Chinese feed and feed dust from bulk grains stored on the ground post-harvest, along with samples from feed mills, personnel and delivery vehicles (Proceedings, 1st International Symposium of prevention of ASF, Henan, China, 2019). To compound this problem, a universally validated method to test feed is not available and routine surveillance testing of feed and feed ingredients is not permitted in the United States. Furthermore, there are no feed additives that are currently approved by the US Food and Drug Administration to mitigate the risk of viral-contaminated feed. Fortunately, research is ongoing to identify additional mitigant candidates and conversations are underway between feed companies and government officials regarding the approval process.
In closing, in a few short years, global agriculture has come a long way in recognizing and accepting the risk of feed and feed ingredients as vehicles for the domestic and transboundary spread of diseases, based on the research efforts cited in this writing. It is hoped that these efforts will continue to stimulate communication and collaboration between the feed and livestock industries, resulting in further research into the emerging concept of 'global feed biosecurity'. Ideally, current and future information regarding the risk of pathogen spread in feed will enhance the accuracy of risk assessments, drive the continual development of efficacious feed-based mitigation strategies and ultimately change the philosophy regarding the global trade of feed ingredients from one that based on price to one where the biosecurity of the feed supply chain is prioritized.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
No ethical approval was required as this is a review article with no original research data.

DATA AVA I L A B I L I T Y S TAT E M E N T
All references used to write this review were disclosed.