Research Results

Sampling Validation for Detecting Swine Viruses in Bulk Ingredients

Project #: 18-24

 Principal Investigators: Dr. Cassandra Jones, et. al.

Institution: Kansas State University

Industry Summary:

Animal feed and ingredients have the potential to transmit swine viruses. Viruses may not be evenly distributed throughout a batch of ingredients or feed, so it is difficult to properly collect a representative sample for analysis, similar to the problem of detecting aflatoxin in a load of corn. The purpose of this experiment was to determine the best type of sample to collect to determine if an ingredient is contaminated with porcine epidemic diarrhea virus (PEDV). To address this objective, we first contaminated soybean meal with two levels of PEDV (Low: 103 TCID50 per gram vs. High: 105 TCID50 per gram) or confirmed it to be PEDV negative. One hundred grams of PEDV-negative soybean meal was added to each of 13 1 kilogram polyethylene tote bags. Next, three grams of PEDV-negative soybean meal (one replicate), low PEDV soybean meal (six replicates), or high PEDV soybean meal (six replicates) was added to the corner of each tote. Finally, 887 grams of PEDV-negative soybean meal was added to each tote, bringing the final mass to 1,000 grams. A cotton gauze swab was wiped across the exterior and top of the polyethylene material. Ten individual samples were collected from each tote using aseptic probes. One gram of each probe sample was reserved for analysis, while one gram from each of the 10 probes per tote were combined to make a composite sample representing each tote. This resulted in each tote having one environmental swab, 10 probe samples, and one composite sample, which were analyzed for PEDV via qRT-PCR. As expected, no PEDV was detected from the swab, probe, or composite sample of the control samples. At the low dose, no PEDV was detected in swabs, individual probes, or composite samples, but was confirmed in 100% of the inoculant samples. At the high dose, only 37% of the probes and 33% (39.2) of the swabs had detectable PEDV. In summary, sampling bulk feed or ingredients for PEDV should include compositing at least 10 individual samples. Clearly, additional research is needed to better detect PEDV in soybean meal and environmental probes when there are low levels of contamination. Therefore, future research efforts should identify alternative methods that have a similar sensitivity to detecting PEDV, but require less time and effort to collect such a sample.

Assessing Tools for the Mitigation of Foreign Animal Disease Introduction and Transmission in Feed

Project #: 17-189

 Principal Investigators: Megan Niederwerder, DVM, PhD

Institution: Kansas State University

Industry Summary:
The risk of feed as a route for introduction and transmission of transboundary animal diseases has been recognized since the 2013 introduction of porcine epidemic diarrhea virus (PEDV). Understanding the risk and mitigation of other foreign animal diseases such as African swine fever virus (ASFV), classical swine fever virus (CSFV) and pseudorabies virus (PRV) in feed and feed ingredients is critical to protecting the health of the US swine herd. The focus of SHIC 17-189 was to 1) identify animal feed ingredients which support survival of CSFV and PRV exposed to transpacific shipment conditions, 2) improve the half-life calculations of ASFV in feed ingredients exposed to transatlantic shipment conditions, and 3) investigate antiviral chemical mitigants as a tool for reducing the risk of introduction and transmission of CSFV, ASFV and PRV in feed and feed ingredients. First, animal feed ingredients were contaminated with CSFV or Chinese PRV and subjected to varying temperature and humidity conditions to simulate transpacific shipment over a 37-day period.  Feed ingredients were then tested for virus at the conclusion of the model by PCR, VI, ± pig bioassay. Second, ASFV was quantified in animal feed ingredients at several time points throughout a 30-day model simulating transatlantic shipment.  Improved ASFV half-life estimates in feed ingredients were calculated to include both standard error and 95% confidence intervals. Third, antiviral chemical mitigants were tested for efficacy against foreign animal diseases in 1) in vitro cell culture models and 2) transboundary shipping models. Overall, this research has improved our ability to quantify risk of CSFV and PRV in feed, implement science-based storage times for ASFV in feed, and identify effective feed additives for risk mitigation of foreign animal diseases through feed.

We confirmed that ASFV Georgia 07 is capable of surviving transoceanic shipment conditions in conventional soybean meal, organic soybean meal, soy oil cake, choline, pet foods, pork sausage casings, and complete feed. These transoceanic conditions have RH values that fluctuate between 20 and 100%, and temperatures between 32 and 78 degrees F. The half-lives of ASFV in all feed ingredients range between 9.6 and 14.2 days with 12.2 days being the average. Implementing storage times for feed ingredients will help reduce the transmission risk.

We have also determined that both CSFV and PRV survive in feed ingredients subjected to a 37-day transoceanic shipment (unpublished). PRV is more stable (similar to ASFV) whereas CSFV is less stable. We are currently evaluating antiviral chemical mitigants that could be used as feed additives to reduce the risk of ASFV transmission in feed.

Validation of a FMDV 3ABC Indirect ELISA (iELISA) for Swine Oral Fluid Specimens

Project #: 18-197

 Investigators: K. Poonsuk, DVM PhD, J. Zimmerman, DVM PhD, L. Giménez-Lirola, PhD

Collaborators: C. Nfon, DVM PhD, A. Clavijo, DVM PhD (National Centres for Animal Diseases – Canada)

Institution: Iowa State University

Foot-and-mouth disease virus (FMDV) remains uncontrolled in most of the world, with circulation of multiple serotypes in endemic areas. Actually, North America is among the few “FMDV-free without vaccination” areas of the world. The current massive level of global trade and traffic means that FADs anywhere in the world present a credible risk to U.S. agriculture. Our recent experience with PEDV is witness to that fact.

Preparing an effective response to the introduction of FMDV is the responsible thing to do. In the event of an FMDV outbreak in North America, effective control and elimination will require rapid detection. In turn, rapid detection will rely on an (1) efficient surveillance sampling technology and (2) immediate access to accurate diagnostic assays. Therefore, the long-term objective of this project is to create a FMD 3ABC antibody indirect ELISA (iELISA) for use with swine oral fluids.

In this study, prototype serum and oral fluid FMDV 3ABC ELISAs were developed using samples from animals of precisely known FMDV status. The optimized tests detected specific antibody in serum and oral fluid samples from FMDV-infected or FMDV-vaccinated pigs by 7-14 days post exposure. Importantly, (1) the response is not serotype specific, i.e., the 3ABC ELISAs detect antibody in animals exposed to serotypes O, A, SAT2, and Asia 1 and (2) the assays detects antibody against a non-structural protein which is not present in FMDV inactivated vaccines, i.e., the test provides for differentiation of vaccinated vs infected animals (DIVA).

Diagnostic testing of swine oral fluid samples has proven to be an effective and reliable method for the surveillance of endemic infectious diseases. Expanding this methodology to include FMDV will help provide FMDV-infected countries a new tool to control the infection and prepare the U.S. industry for a “worst-case” scenario.

Case Study: Is it Time for an NPIP Like Program for the US Pork Industry?

Project #: 18-189

Principle Investigator: Rodger Main

 Co-investigators: Pam Zaabel, Kerry Leedom-Larson, James Roth, and Jeff Zimmerman.

Institution: Iowa State University

A study was commissioned in 2018 with the aim of seeking a more in-depth understanding of the National Poultry Improvement Plan (NPIP) and assessing the potential for an NPIP like program to support the US pork industry. NPIP is a unique industry, state, and federal partnership that has long served to safeguard, improve, and assure the health of US poultry and enhance the competitiveness of the US poultry and egg industries in the domestic and global marketplace. Participation in NPIP is voluntary and almost universal among commercial poultry and egg operations throughout the US. Participants utilize NPIP to certify the health status of US poultry and egg flocks, hatcheries, slaughter plants, products, and states in accordance with NPIP’s officially recognized standards and definitions. NPIP’s health status certifications are used to demonstrate evidence of freedom of both trade and non-trade impacting diseases of poultry. NPIP is a working and active system of animal health control whose programs content and direction are informed and updated every two years by a formal congress of industry stakeholders and subject matter experts. NPIP’s program definitions, standards, and health status classifications are broadly recognized across all 50 states and by international trading partners. While participation in NPIP is voluntary, specified NPIP health status certifications are commonly required at points of sale, exhibition, and for interstate and international commerce. NPIP’s Avian Influenza Virus (AIV) surveillance programs and health status certifications held by meat-type chicken and turkey slaughter plants, commercial table egg laying operations, and states have played a primary role in helping sustain export markets and interstate commerce from unaffected regions during times of an AIV outbreak of significance affecting US commercial poultry operations.

Development and Evaluation of a Dual Matrix Derum/Oral Gluid Atypical Porcine Pestivirus ELISA Using Known Status Samples

Project #: 18-136

Principle Investigator: Bailey Arruda

Institution: Iowa State University

Industry Summary:

Atypical porcine pestivirus (APPV) is the most common cause of congenital tremor (CT) in pigs. CT is a disease of neonatal pigs that is characterized by bilateral, clonic contractions of skeletal muscle that are seen within hours of birth and stop when piglets are at rest. APPV is transmitted from the dam to fetuses resulting in all or a subset of piglets with CT. Gilt and low parity sows are frequently reported to have CT litters; however, CT litters can be seen with multiparous sows as well. Outbreaks of CT litters, reports of up to 50% of litters affected, are most commonly observed in herds that are newly stocked, have changed genetics or have a high gilt replacement rate. In such outbreaks, it is not uncommon to have litter mortality reported to be 30 to 40%. Regardless of parity, CT litters are likely due to insufficient dam immunity at a critical timeframe of gestation. It is not currently known the most common route of exposure and infection in dams. Semen as well as cohorts may be involved.

There is limited information concerning the ecology, epidemiology and pathophysiology of APPV. Currently, there is no available serologic assay to evaluate the immunity of a dam or herd. Such an assay would provide meaningful information to assess the effectiveness of preventative measures such as acclimation and vaccination as well as improve our understating of the infection dynamics and herd impact of APPV.

To address this need, a panel of specimens including serum and oral fluid was generated that contained polyclonal antibodies against APPV. To achieve this objective, twenty two cesarean derived colostrum deprived (CDCD), crossbred, mixed-sex, 6-week-old pigs were individually identified, blocked by litter and randomly assigned to one of two groups in separate rooms based on inoculum and pen. Two pigs were placed per pen with three pens (negative control animals) or eight pens (positive control animals) per room. Oral fluids, serum, and nasal swab samples were collected prior to challenge and submitted for detection APPV by PCR prior to inoculation. Pigs were inoculated with MEM (n=6; negative control) or APPV (n=16) intravenously, intramuscularly and intranasally. Serum, pen-based oral fluids samples (3 or 8 pens/group), and individual nasal swabs were collected 0, 3, 7, 10, 14, 17, 21, 28, 35, 42, 49, 56, 63, and 70 days post inoculation (DPI). Oral fluids were also collected 31, 38, 45, 52, 59 and 66 DPI.

APPV was first detected at 10 DPI in the serum of four inoculated animals. By 35 DPI, APPV was detected in the serum of every APPV-inoculated animal and all animals remained positive until the end of the study at 70 DPI. APPV was detected in 63% and 83% of oral fluids at 14 and 17 DPI, respectively. By 24 DPI, all oral fluid samples were positive and remained positive until the end of the study at low Cq values (17.3-20.0). APPV was first detected in nasal swabs from four animals at 17 DPI with nasal shedding detected in animals at 42, 49, and 56 DPI. APPV was not detected in any sample type from negative control pigs.

With the panel of known status specimens generated at the completion of the first objective, two Erns iELISA assays (serum and oral fluid) were developed using a selected immunogenic region of Erns which was cloned, expressed and purified as a recombinant polypeptide. The serum and oral fluid assays have a 100% diagnostic specificity at a cut-off ≥0.10 and 0.15, respectively.

This is the first study to experimentally infect swine with APPV and monitor the infection dynamics overtime out to 70 DPI. The results of this experimental inoculation provide evidence that APPV viremia can be prolonged, at least 60 days could be expected following intentional exposure. These finding may be applicable if gilt acclimation protocols are undertaken. Additionally, based on the results of this study it appears that oral fluid is an appropriate and likely highly sensitive sample type to monitor herd status by RT-qPCR. Lastly, both oral fluid and serum iELISA assays can be used to evaluate individual and herd status prior to and following intervention strategies. This project provided important foundational knowledge concerning the infection dynamics of APPV in experimentally infected swine while also providing the necessary samples to develop and evaluate serologic assays that will assist in furthering our understanding APPV and preventing CT litters.

Development and Evaluation of a Real-Time RT-PCR and a Field-Deployable RT-Insulated Isothermal PCR for the Detection of Seneca Valley Virus

Project #: 16-271

Investigators:Jianqiang Zhang, Charles Nfon, Chuan-Fu Tsai, Chien-Hsien Lee, Lindsay Fredericks, Qi Chen,
Avanti Sinha, Sarah Bade, Karen Harmon, Pablo Piñeyro, Phillip Gauger, Yun-Long Tsai,
Hwa-Tang Thomas Wang and Pei-Yu Alison Lee

Institution: BMC Veterinary Research

Industry Summary:

Seneca Valley virus (SVV) has emerged in multiple countries in recent years. SVV infection can cause vesicular lesions clinically indistinguishable from those caused by other vesicular disease viruses, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV). Sensitive and specific RT-PCR assays for the SVV detection is necessary for differential diagnosis. Real-time RT-PCR (rRT-PCR) has been used for the detection of many RNA viruses. The insulated isothermal PCR (iiPCR) on a portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, SVV rRT-PCR and RT-iiPCR were developed and validated.

Characterization of United States Breeding-Stock Companies International Transport Biosecurity Practices

Project #: 18-193

Investigator: Cesar A Corzo

Institution: University of Minnesota

Industry Summary:

Pig transport within and between countries continue to play an important role for production companies. These companies are always seeking the highest level of genetic makeup on their market pigs aiming at improving whole system economics and maintaining their competitiveness; therefore, investments into obtaining the highest genetic level involve import/export of breeding-stock animals. However, transport of livestock around the globe may represent risk as infected animals may harbor exotic pathogens that could be introduced into another country. Data on the frequency, quantity and type of pigs entering and exiting the United States is scarce. In addition, the process by which these exports/imports occurs is not well known; therefore, further understanding of the frequency of these events and procedures are warranted in order to understand whether these are risky events.

Objectives: 1) Determine the frequency of international breeding-stock exports or imports and their country of destination and/or origin; and, 2) To characterize the procedures currently implemented by breeding-stock companies during the export or import processes.
Methods: For objective 1, databases containing information related to breeding-stock imports/exports were obtained for analysis. More specifically, data sources were divided in two main groups, public databases and breeding-stock private databases. Public databases were identified through website searches whereas private databases were requested directly from individual breeding-stock companies by an invitation to participate in this project. For objective 2, participating breeding-stock companies were asked to share their transport protocols together with the possibility of us witnessing their import/export process.

Results: Based on official USDA records, between 2007 and 2018, a total of 839,152 pigs (e.g gilts or boars) were imported into the United States. Most of these pigs originated from Canada, while less than 3% were imported from Western Europe. On the other hand, breeding pig exports accounted for 382,118 pigs between 2007 and 2018 with Asia being the main destination (54.0%), followed by Mexico (31.3%) and South America (5.7%). A total of 8 breeding-stock companies were invited to participate in this study, 50% of these accepted the invitation by sharing import/export protocols. Biosecurity procedures are across companies are similar which assure the maintenance of the health status of these pigs.
Implications: The results of this study show that exports/imports are a frequent event. Imports from outside North America occurs less frequently.

Development and Validation of ELISA to Detect IgA, IgM, IgG in Serum and Oral Fluids to Porcine Teschovirus (PTV)

Project #: 17-199

Investigator: Douglas Marthaler

Institution: Kansas State University

Industry Summary:

Belonging to the family Picornaviridae, Porcine teschovirus (PTV) was first identified in pigs exhibiting various symptoms including ataxia, nystagmus, convulsions, polioencephalomyelitis, and paralysis. While previous molecular methods are available to detect PTV, the lack of pan-PTV ELISA (ability to detect all the serotypes) hampers our ability to conduct PTV surveillance and determination of the immune status within a herd. The aimed of our study was to develop and validate a pan-PTV indirect ELISA for diagnostics.

The VP1 protein of picornaviruses stimulates the strongest immune response but is also serotype specific. Thus, we created three protein constructs VP1, VP2, and VP2-VP1 linker. The VP2-VP1 linker protein generated the highest fluorescence levels for detection. The diagnostic sensitivity and specificity was determined using the known PTV antibody status of 558 serum samples (369 positive and 189 negatives). A cutoff of 0.4105 was used, yielding a diagnostic sensitivity and specificity of 99.2% and 98.4%, respectively. Using a single lot of internal control serum, PTV ELISA exhibited a within-plate Coefficient of Variation (CV) of 7.32%, within-run CV of 7.85%, and between-runs CV of 9.24%, indicating the highly repeatable with serum samples.

The indirect PTV ELISA was employed to investigate the immune response in post-weaned piglets after PTV exposure. During the first week post-weaning, the PTV antibody response was negative. On day 35, the ELISA detected PTV antibodies, and the detection of antibodies peaked on the last day of the study (63 days). Fluorescent microsphere immunoassay (FMIA) on the BioRad system was used to detect PTV antibodies in oral fluids. However, the ability to detect PTV antibodies in oral fluids was highly variable. Between days 35 and 41, the ability to detect PTV antibodies was sporadic, and PTV antibodies were not detected after 41 days of age. These results suggest oral fluids may not be a suitable specimen to determine the PTV immune status of a herd.

In conclusion, we developed and validated an indirect PTV ELISA for use in diagnostics. In addition, we measure the antibody response to PTV in weaned piglets and determined the PTV antibodies in oral fluids was variable.

Developing Surveillance Systems for Emerging and Foreign Animal Diseases of Swine

Project #: 17-141

Investigator: Jeff Zimmerman, DVM PhD

Institution: Iowa State University

Industry Summary:

Effective surveillance should efficiently collect data for production and/or business planning, document freedom from specific pathogens, and guide a rapid, effective response to emerging and/or FADs. Current on-farm or regional surveillance programs routinely fail to meet these targets. In part, this is because the industry has changed over time and no longer conforms to the assumptions under which our surveillance systems were originally designed. As a result, surveillance either is not done or is done ineffectively.

On-farm surveillance The statistical theory on which on-farm surveillance was originally based assumes: (1) subjects (pigs) are independent, (2) all pigs have an equal probability of being selected for sampling, and (3) the farm has a stable, homogenous pig population. Traditional farms fit these assumptions – hence the “30 sample” approach worked in the PRV eradication program – but current swine production systems do not.

Contemporary production systems differ from traditional farms in ways that are incompatible with traditional surveillance: (1) Today’s production systems are much larger than in the past. Iowa farms averaged a total inventory of 250 animals in 1980 (Flora et al., 2007) versus 3,265 according to a study commissioned by the Iowa Pork Producers Association in 2016 (https://www.iowapork.org/study-iowa-pork-industry-remains-important-economic-driver/). (2) Pigs no longer run free in pastures or feedlots. Instead, management of large swine populations requires physical segregation by age and stage into buildings and pens. (3) Swine populations on modern farms experience rapid turnover of animals and frequent introduction of new animals – often of a different disease status. Thus, current production systems rely on extensive movement of pigs, people, trucks, and feedstuffs between sites. This connects distant places/populations and facilitates the rapid movement of pathogens between them.

Surveillance at the farm level In NPB #13-157 (Rotolo et al., 2017), we showed that disease on contemporary farms moved in a spatiotemporal fashion (non-random). This led us to develop new surveillance guidelines for on-farm surveillance based on spatial (non-random) sampling. This “fixed spatial sampling” approach is being used in the U.S. and elsewhere.

Surveillance at a regional level Efficient regional surveillance is fundamental to detecting the incursion of new pathogens and in monitoring regional disease control/elimination projects. Thus, the current project moved surveillance to the regional level with the objective of developing more efficient regional surveillance methods (fewer samples, but better detection). In this project, we tested the hypothesis that disease exhibited a spatiotemporal pattern of spread at the regional level (just as we saw on farms). The emergence of PEDV in April 2013 provided the opportunity to examine this question.

Using PEDV testing results from the Iowa State University Veterinary Diagnostic Laboratory (at the county level to protect client confidentiality), we found a spatiotemporal pattern of PEDV spread. This means that, just as for on-farm sampling, the assumptions upon which regional surveillance have been based do not hold in today’s world. This is important because it means that new guidelines for regional surveillance should be developed using statistically-appropriate modelling to account for the spatial and temporal correlation in disease spread. As a first effort in developing new guidelines, we have shown that spatially balanced sampling through generalized random–tessellation stratified (GRTS) gives a higher power of detection than traditional simple random sampling (SRS) using simulation studies mimicking real PEDV data.

Thus, our research has provided a better understanding of the spatiotemporal nature of disease spread. Initial assessment showed that use of a spatially balanced sampling scheme improved the power of disease detection and the efficiency of the disease surveillance.

Pilot Study to Evaluate the Use of a Fluorescent Powder (Glo Germ) to Study the Transfer of Contamination From Livestock Trailers to the Center Alleyway and Pens in the Barn During Marketing Events

Project #: 19-147

Investigators: Chelsea Ruston, DVM, Daniel Linhares, DVM, PhD, Pete Thomas, DVM, Derald J. Holtkamp, DVM, MS

Institutions: Iowa State University College of Veterinary Medicine, Ames, Iowa,  Iowa Select Farms, Iowa Falls, Iowa.

Industry Summary:

Currently, many livestock trailers in the United States are not washed, disinfected or dried between loads of market pigs due to the lack of trailers, truck washes and other swine transport related infrastructure. If livestock trailers or other carrying agents associated with the marketing event become contaminated, it is unlikely that the contamination is mitigated unless specific procedures, such as washing are performed. Under these circumstances, the livestock trailer, truck and driver returning directly from a swine slaughter plant are likely frequently contaminated with live infectious PRRSv or PEDv or both when they enter a growing pig site to haul the next load.

Half-Life for Feed Holding Time

Project #: 18-211

Investigators: Diego G. Diel

Institution: South Dakota State University

Industry Summary:

This study evaluated the stability of Senecavirus A, a picornavirus surrogate for foot-and-mouth disease virus (FMDV), and a pathogen that is known to survive for prolonged time in several swine feed ingredients. Common swine feed ingredients including conventional soybean meal (SBM-C), DDGS, lysine and Vitamin D were inoculated with a constant dose of SVA and incubated under different temperatures (4oC [39.6oF], 15oC [59oF] and 30oC [86oF]) to assess the effect of temperature on the stability of the virus. Samples incubated at each temperature were collected weekly for 14 weeks (days 1 through 91) and the amount of viable SVA was determined by virus titrations in the laboratory. Control samples consisted of stock virus incubated in a plastic container without a feed ingredient. The control samples were included in all temperatures tested, collected and processed following the same sample schedule as above. SVA was inactivated within 7-14 days when incubated at 30oC (86oF). Lower incubation temperatures (4oC [39.6oF], 15oC [59oF]), however, favored survival of SVA for 28 or up to 91 days, respectively. The results from this study demonstrate that SBM and DDGS provide a good matrix for the survival of SVA. Lysine and vitamin D, on the other hand only supported SVA survival for 21 days, even when incubated at lower more favorable temperatures (4oC [39.6oF]). A clear effect of temperature on the stability of the SVA was also observed. When SVA spiked-SBM or -DDGS were incubated at 4oC, infectious SVA was recovered from these samples until the end of the experiment on week 14 or day 91 post-incubation. It is important to point out that SVA viability decayed much faster (7-21 days) in control samples, in which the virus stock was deposited directly in a plastic tube without a feed matrix. The half-life, or the time required for infectious SVA amounts to decrease by one-half, were also determined in all feed ingredients. These results, consistent with the decay rate, show an extended half-life for SVA in SBM and DDGS when incubated at 4oC (10.9 and 37.9 days, respectively). Incubation at higher temperatures results in rapid degradation of the virus and very short half-life’s (1.25 and 1.36 days for SBM and DDGS, for example). In conclusion, results from these studies confirm that common swine feed ingredients such as SBM and DDGS provide a good environment for virus survival, increasing the overall stability of SVA, an important swine pathogen and surrogate for FMDV to survive for long periods of time. A clear effect of temperature was observed, with higher environmental temperatures resulting in rapid virus decay even in the most favorable feed ingredients. These results may help the swine industry to devise mitigation strategies that consider holding times for feed ingredients that are imported from countries where foreign animal diseases are endemic.

Evaluation of Chemical Mitigants for Neutralizing the Risk of Foreign Animal Diseases in Contaminated Feed Ingredient

Project #: 17-187

Investigators: Diego G. Diel and Scott Dee

Institution: South Dakota State University

Industry Summary:

The North American swine industry is under constant threat of foreign animal disease (FAD) entry. The goal of this study was to identify chemical feed additives that could be used to mitigate the risk of pathogen transmission through feed. Based on the outcome of our previous feed survival study, “high-risk” combinations of viruses and ingredients were identified. Ten mitigant candidates were selected and screened against the target pathogens. “High risk” combinations of virus and ingredient that were tested include: Senecavirus A (SVA; Soybean meal, lysine, choline and vitamin D); Porcine epidemic diarrhea virus (PEDV; Soybean meal, lysine, choline and vitamin D); Porcine reproductive and respiratory syndrome virus (PRRSV; Soybean meal and DDGS); and, Bovine herpesvirus type 1 (BoHV-1 – surrogate for pseudorabies virus [PRV]; soybean meal and soy oil cake). Results from our study show that among the 10 feed additives tested, a select group of additives presented promising efficacy against target swine pathogens. Although none of the feed additives tested completely inactivated the pathogen(s), consistent reductions in viral titers were observed when a select group of mitigants was used (KANA102 and MCFA). Interestingly, these two products showed promising results for all four viruses tested (SVA, PEDV, PRRSV and BoHV-1). Another important observation of our study is the fact that both KANA102 and MCFA are based on a blend of medium chain fatty acids. In addition to MCFA based products, Activate DA a blend of organic acids and a methionine analogue was also effective against most pathogens screened in our study. These results demonstrate that a select group of feed additives have the potential to be used as chemical mitigants to reduce viral contamination levels in feed. Further studies are warranted to assess the mechanism of action of those products and to assess their efficacy following natural ingestion of contaminated and mitigated feed.

Development and Validation of a Scoring System to Assess the Relative Vulnerability of Swine Breeding Herds to the Introduction of PRRS Virus

Project #:

Investigators: Gustavo S. Silva, Luis G. Corbellini, Daniel L.C. Linharea, Kimberlee L. Baker, Derald J. Holtkamp

Institution: Iowa State University and Universidade Federal do Rio Grande do Sul

Industry Summary:

In modern veterinary practice, disease prevention in livestock populations has become increasingly more important (Kimman et al., 2013). This change in focus includes the adoption of biosecurity practices, which are defined as “the implementation of practices that reduce the risk of disease agents being introduced and spread into a population” (Food and Agriculture Organization, 2010).

Previous studies have demonstrated the effect of biosecurity on prevention or reduction of disease incidence (Alonso et al., 2013; Amass, 2004; Hagenaars, 2008). However, evaluation of biosecurity practices on pig farms is extremely complex. Pathogens can be introduced into pig farms in different ways (Pileri and Mateu, 2016) and the effectiveness of specific biosecurity practices depends on the characteristics of the herd, characteristics of the premises, and surrounding areas and connections to other swine premises. Porcine reproductive and respiratory syndrome (PRRS) continues to be a major health challenge in U.S. herds since it was first reported in 1989 (Keffaber, 1989). While the incidence in the U.S. has declined in recent years (Morrison et al., 2015), the prevalence continues to increase over time (MSHMP, 2018) and PRRS virus (PRRSv) still causes significant economic losses worldwide (Holtkamp et al., 2013; Nathues et al., 2017). PRRSv can be transmitted between farms via different risk events including swine movements, pickup and deliveries of supplies from or to farms, people movement, contact with other animals, air and water (Otake et al., 2002; Perez et al., 2015; Zimmerman et al., 2012).

Herd-specific biosecurity assessments are useful to determine how PRRSv may be introduced in swine herds and research is needed to quantify the relative importance of specific biosecurity practices to reduce the frequency of outbreaks. Biosecurity assessments have been used to identify relevant risk factors of disease spread onto swine farms (Bottoms et al., 2013; Holtkamp et al., 2011; Laanen et al., 2013; Sternberg Lewerin et al., 2015). However, identifying the vulnerabilities to PRRSv introduction specific to a certain production system and developing a generalized score that accounts for all major risk events is an intrinsically complex process.

Given the complexity of evaluating biosecurity practices to prevent the introduction of PRRSv, applying a technique that uses multiple factors to score swine breeding herds based on their relative vulnerability to PRRSv introduction would be beneficial for prioritizing and identifying gaps in biosecurity practices and predicting the frequency of outbreaks. Several methods exist to evaluate these factors, allowing for a ranking of specific factors by relative importance. One method by which to do this is multi-criteria decision analyses (MCDA) (Belton and Stewar, 2002), which has been applied extensively in a variety of fields (Santos et al., 2017; Steele et al., 2009; Thokala et al., 2016), including to assess vulnerability (Cardona, 2003; Joerin et al., 2010). MCDA was chosen for the present study because it provides a systematic way to integrate information from a range of sources, compare scenarios and prioritize decisions (Cox et al., 2013).

The objective of this study was to develop a biosecurity vulnerability score (BVS) that represents the relative vulnerability of swine breeding herds to the introduction of PRRSv. To validate the BVS, a survey of biosecurity practices and PRRS outbreak histories in 125 breed-to-wean herds in two different populations in the U.S. was used. Data on the frequency of PRRS outbreaks was used to test the hypothesis that BVS were different between farms that have a low incidence of PRRS outbreaks, compared to farms that have a high incidence.

Validation of a Low-cost Tool for Senecavirus A Detection, and Surveillance of Viral Prevalence in United States Feed Mills

Project #: 17-188

Principal Investigator: Cassandra Jones

Institution: Kansas State University

Industry Summary:

Senecavirus A (SVA), previously known as Seneca Valley Virus, is a detrimental pathogen in the United States swine industry. Transmission is not well understood, but its similarity to foot and mouth disease virus (FMDV) suggests direct contact with people or fomites may spread the virus. Once present, viruses in feed, feed ingredients, and feed mills are difficult to mitigate. While contaminated surfaces in a feed mill have been demonstrated as a potential vector for bacterial and viral transmission, there is currently no approved method for its evaluation of viral contamination. Therefore, the objective of this Experiment 1 was to validate standardized swabbing techniques for detection of SVA. A secondary objective was to determine if a freeze/thaw cycle impacted detectable RNA. This experiment included 3 forms (inoculum, feed, or swab), 4 doses of SVA (none, low, medium, or high), and 2 storage methods (analyzed initially vs. after a freeze/thaw cycle). The SVA was added to swine feed, with 1 g reserved, and the remaining spread over a stainless steel coupon. Feed was removed, but residual feed dust remained. Next, surfaces were swabbed and samples split, with one set analyzed initially, and another frozen for 7 days, then thawed and analyzed. Results are reported as the quantity of detectable SVA as determined by threshold cycle (Ct) in qRT-PCR, where the higher the Ct, the less detectable virus was identified. The results demonstrate that sample type impacted the quantity of detectable SVA, where feed samples were approximately 8 Ct higher than the inoculum, and swab samples were approximately 4 Ct higher than feed. A freeze/thaw cycle did not impact detectable SVA compared to samples that were analyzed immediately.

In Experiment 2, the objective was to determine the prevalence and distribution of SVA in United States swine feed mills as an indicator of risk of domestic and foreign animal disease transmission through feed. A total of 375 samples were collected from 11 surfaces + one feed sample collected from 11 different feed mills manufacturing swine feed located in 8 different states. Feed mills include 5 producing both mash and pelleted feed in KS, CO, OK, NC, and IA, and 6 producing only mash feed in KS, NC, MN, IA, IN, and IL. Within a mill, locations included ingredient pit grating, fat intake inlets, exterior of pellet mill (only in feed mills with pelleting capacity), finished product boot bin, load-out auger, finished feed, floor dust in the break/control room, floor dust in receiving, floor dust in the manufacturing area, floor dust in the warehouse, worker shoe bottoms, and broom in the manufacturing area. To account for potential seasonality associated with pathogenic hazards, the same locations in feed mills were swabbed in Late Fall 2016, Winter 2016/17, and Summer 2017. Notably, no mills were manufacturing feed for SVA-positive herds at the time of analysis. Five of 375 samples analyzed positive for SVA, with Ct ranging from 37.4 to 39.9. One positive sample was collected in late Fall, while the other four positive samples were collected in Winter. No positive samples were identified in Summer. Two samples were from load-out augers, and one each from fat intake inlet, floor dust in the receiving area, and worker shoes. A sow farm being fed by the mill with SVA on worker shoes was subsequently diagnosed with SVA after the sample as collected.

These results indicate that an environmental swab can be used to detect SVA in feed, however with approximately 4 Ct less precision than analyzing feed samples directly. Furthermore, the limit of detection of SVA in environmental swabs appears to be near 10^3 TCID50/mL. Samples can be frozen prior to analysis without impacting detectable SVA RNA. Finally, SVA was not widespread throughout the swine feed mills analyzed in this experiment, but its presence in a mill may be indicative of disease risk or entry into pig populations, particularly through worker shoes.

Development of a FMDV 3ABC Antibody ELISA for Swine Oral Fluid Specimens

Project #: 17-191

Investigators: J. Zimmerman, DVM PhD, L. Giménez-Lirola, PhD, K. Poonsuk, DVM PhD.

Institution: Iowa State University

Industry Summary:

Foot-and-mouth disease virus (FMDV) remains uncontrolled in most of the world, with circulation of multiple serotypes in endemic areas.  Actually, North America is among the few “FMDV-free without vaccination” areas of the world.  The current massive level of global trade and traffic means that FADs anywhere in the world present a credible risk to U.S. agriculture.  Our recent experience with PEDV is witness to that fact.

Development and Validation of a Single-tube,Triplex RT-PCR Assay for Differential Detection of Highly Virulent Chinese Strains of Pseudorabies Virus

Project #: 16-250

Principal Investigator: Aruna Ambagala

Institution: National Centre for Foreign Animal Disease- Winnipeg, MB

Collaborators: Guang-Zhi Tong, En-Min Zhou, Jianfa Bai, Lalitha Peddireddi, John Schiltz, Sabrina Swenson, Karthik K Shanmuganatham

Industry Summary:

Pseudorabies virus (PRV) causes pseudorabies or Aujeszky’s disease in livestock and wild mammals; however pigs are the main host and reservoir for this virus.  It causes deadly disease in newborn piglets, respiratory problems in growing and fattening pigs, and reproductive problems in pregnant sows. Like other herpesviruses, PRV establishes a lifelong infection in the nervous system followed by subsequent shedding of infectious virus. Pseudorabies has spread throughout the world, but Canada, Greenland, and Australia are considered free of this disease. In 2004, PRV was eliminated from the US commercial swine herds, but the virus remains in some localized feral swine populations.  China is considered the largest pork producer in the world.  The earliest documented PRV outbreak in China was in 1947.  Since 1990s, more than 80% of pigs in China have been vaccinated and the clinical disease was well controlled. In late 2011 however, a newly PRV virus (variant) which cause severe disease surfaced in PRV vaccinated pig herds in Northern China. Since then, this virus has spread across China causing severe economic losses.

North American Domestic Pigs are Susceptible to Experimental Infection with Japanese Encephalitis Virus

Project #: 16-258

Principal Investigators: So Lee Park, Yan-Jang S. Huang, Amy C. Lyons, Victoria B. Ayers, Susan M. Hettenbach, D. Scott McVey, Kenneth R. Burton, Stephen Higgs, and Dana L. Vanlandingham

Industry Summary:

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is capable of causing encephalitic diseases in children. While humans can succumb to severe disease, the transmission cycle is maintained by viremic birds and pigs in endemic regions. Although JEV is regarded as a significant threat to the United States (U.S.), the susceptibility of domestic swine to JEV infection has not been evaluated. In this study, domestic pigs from North America were intravenously challenged with JEV to characterize the pathological outcomes. Systemic infection followed by the development of neutralizing antibodies were observed in all challenged animals. While most clinical signs were limited to nonspecific symptoms, virus dissemination and neuroinvasion was observed at the acute phase of infection. Detection of infectious viruses in nasal secretions suggest infected animals are likely to promote the vector-free transmission of JEV. Viral RNA present in tonsils at 28 days post infection demonstrates the likelihood of persistent infection. In summary, our findings indicate that domestic pigs can potentially become amplification hosts in the event of an introduction of JEV into the U.S. Vector-free transmission to immunologically naïve vertebrate hosts is also likely through nasal shedding of infectious viruses.

Validation of a Real-Time Reverse Transcription PCR Assay for Detection of Porcine Kobuvirus (PKV) in Porcine Diagnostic Samples

Project #: 17-144

Principal Investigators: Dr. Phil Gauger DVM, PhD, Associate Professor; Dr. Karen Harmon, PhD, Clinical Associate Professor

Institution: Iowa State University

Industry Summary:

Porcine kobuvirus (PKV) is an enteric virus detected in swine feces that emerged in the pig population during the previous two decades. Kobuviruses are members of the family Picornaviridae, which is a different virus family compared to PEDV, PDCoV and TGEV or Rotavirus. The first PKV was detected in Europe in 2008 and since have been identified in domestic swine in China, Thailand, Japan, Korea and the United States (US). Porcine kobuviruses have been associated with clinical diarrhea in some swine populations; however, PKV is also detected in feces from healthy swine lacking clinical signs. A study conducted in the US detected PKV in similar numbers of affected and non-affected swine.

The objective of this research was to validate a real-time reverse transcriptase PCR (rRT-PCR) that would detect US strains of PKV in feces, fecal swabs and oral fluids collected from swine. Sequencing assays were developed and validated based on the genes specific to US strains of PKV. In addition, the assay was evaluated in China using clinical samples that contained PKV strains specific to that region.

Porcine feces, fecal swabs and oral fluids were collected from cases submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL). There were 1,845 samples collected at the ISU VDL and evaluated by the rRT-PCR including 738 oral fluids (OF), 579 feces, and 528 fecal swabs. Approximately 85.8% (633/738) of the oral fluids, 54.2% (314/579) of the feces and 71.2% (376/528) of the fecal swabs were considered positive for porcine kobuvirus. Sequencing confirmed the detection of PKV on positive samples. Feces and tissue homogenates from 112 porcine clinical samples were evaluated with the ISU VDL rRT-PCR in China. There were 23 PKV positive samples using the ISU VDL test confirmed by sequencing indicating the ISU VDL test has the ability to detect Chinese strains of PKV.

Collectively, PKV RNA is present and can be detected in porcine fecal samples and oral fluids using an diagnostic tests validated at the ISU VDL. Validated samples at this time is limited to oral fluids, fecal swabs and feces. Sequencing is available and can be used for monitoring different strains of PKV in swine populations. The ISU VDL diagnostic test also successfully detected strains of PKV from other regions suggesting the emergence of PKV from different geographic regions are detectable using this test. Overall, the large number of positive samples suggest PKV is widespread in US swine and further research is needed to learn if pigs with or without diarrhea are infected with PKV or if different strains of the virus are more likely to cause diarrhea in swine.

Contact information: Dr. Phil Gauger, Iowa State University Veterinary Diagnostic Laboratory. 515-294-1950; pcgauger@iastate.edu

Final Report: Description of Biosecurity Aspects of Herds With Low or High PRRS Incidence and Comparison Within and Between Production Systems

Project #: 16-273

Principal Investigator: Daniel Linhares

Co Investigators: Gustavo Silva, Kimberlee Baker, Derald Holtkamp, Bob Morrison

Institution: Iowa State University, University of Minnesota

Industry Summary:

Porcine reproductive and respiratory syndrome (PRRS) compromises the health of millions of pigs and costs the industry $664 million annually. Thus, swine producers adopt biosecurity measures with intent to decrease the frequency of PRRS outbreaks. There is a critical need to better understand the effects of biosecurity aspects on frequency of PRRS outbreaks in breeding herds.
Therefore, the objective of this study was to describe key differences in the biosecurity aspects of breeding herds with relative low PRRS incidence, compared to those with relatively high PRRS incidence.

This study included herds from 14 production systems in the US. Within each production system herd selection was completed by ranking production system’s herds based on the number of PRRS outbreaks since 2013 and then randomly selecting 3 farms from the 25th and 75th percentiles. The farms from 25th percentile were defined as ‘low incidence’, and farms in the 75th percentile defined as ‘high incidence’. The biosecurity aspects of each breeding herd were assessed using a 346 questions biosecurity survey that contained multiple choice and short answer questions about herd demographics, swine density, PRRS outbreak history, frequency of risk events, and biosecurity practices related to swine transport, people movement, carcass disposal, supply deliveries, and other risk events. Statistical methods were used to determine which biosecurity aspects were significantly different between the low and high PRRS incidence farms.

Fourteen herd sets (84 herds) were enrolled in the study representing 13 states. The average herd size was 3,453 breeding females (range: 543-7,200) for the low PRRS incidence group and 4,099 breeding females (range: 1,000-10,852) for the high PRRS incidence group. Four general areas of biosecurity separated the low and high PRRS incidence farms: (1) monthly event frequency, (2) downtime requirements, (3) swine density, and (4) operational connections to other swine sites.
Rendering was the most significant difference between the groups: 64.3% of high PRRS incidence herds used rendering compared to 31% of low PRRS incidence herds. Mean monthly rendering frequency was 12.7 for high PRRS incidence herds and 5.7 for low PRRS incidence herds. High PRRS incidence farms had a higher monthly frequency of visits from visitors (range: 1-24) than low PRRS incidence farms (range: 0.5-8). Low PRRS incidence farms had significantly longer downtime requirements for visitors and manure removal personnel than high PRRS incidence farms. High PRRS incidence farms were located in areas with significantly higher densities of wean-to-finish swine. Interestingly, a higher number of boars and finishing pigs within a 3-mile radius were significantly associated with a low PRRS incidence. This may be accounted for, in part, by the higher level of biosecurity practiced at boar studs. Operational connections to other swine sites were also important as several operational connection related variables were associated with high PRRS incidence.

These observations will enable the swine industry to more effectively allocate resources to specific aspects of biosecurity which may help reduce the animal welfare and economic impacts of PRRS in the future. Our group will continue to develop biosecurity scores that correlate (help to explain) the frequency of outbreaks. In a nutshell, this study demonstrated the importance of number of events on the biosecurity risk. In other words, we encourage producers to evaluate possibility of reducing the number of pig animal movements (e.g. reducing number of weaning events per month), and number of people entry in the farm (e.g. reducing number of re-entry events).

Also, there was a significant variation in number of PRRS outbreaks in breeding herds. The risk of PRRS exposure can be measured using ‘biosecurity scores’ derived from questionnaires. Benchmarking the scores, and simple outcomes such as number of pig movements, and number of people entry/re-entry per 1,000 sows may be a great tool for managers and producers to identify opportunities to reduce the vulnerability of their swine operations.

Development of Sensitive and Reliable Diagnostic Assay to Detect Atypical Procine Pestivirus (APPV) in Swine

Project #: 16-256

Principal Investigator: Lalitha Peddireddi

Institution: Kansas State Veterinary Diagnostic Laboratory, Kansas State University

Industry Summary:

Atypical Porcine Pestivirus (APPV) is reported as an etiological agent for type A-II congenital tremors in newborn piglets. Since the first report of APPV from US in 2015, there have been several reports of this virus from around the world. APPV strains reported thus far from different parts of the world exhibit significant genetic diversity (7-17%). Currently used PCR-based APPV detection methods were developed based on limited sequencing information available at the time of their design and are primarily used for research purposes. Therefore, a well validated, highly sensitive and reliable diagnostic assay, developed based on the most up-to-date sequence information, is critical for effective detection of all the genetic variants of APPV. The main aim of this study is to develop a real-time RT-PCR (qRT-PCR) assay, capable of detecting all currently known genetically divergent APPV strains, and fully validate its use in diagnosing APPV infections in the US swine herds. To achieve the main goal of this project, our objectives included, compiling all newly available APPV sequences, generating more complete genome sequences by sequencing of at least 20 APPV positive clinical samples, from different geographical regions, and use the most updated sequence information to design and validate a new qRT-PCR assay. At the time of our initial assay design, there were a total of 7 published full genome sequences available and our sequencing efforts resulted in two complete genomes out of 20 APPV positive clinical samples. So, our initial assay design targeting a highly conserved region in NA5B gene was based of 9 full genomes and 56 partial sequences. After the availability of 4 additional full genome APPV sequences from China, we noted primer-template mismatches within the NS5B target primers as the China strains exhibited high sequence variability (~17%) compared to other APPV strains reported to date.  To overcome potential limitations of this assays ability to detect highly divergent China strains, we have modified our assay design to include a second assay targeting a highly conserved region in NS3 gene as an additional target. So, our final assay is a triplex assay with two APPV target regions (NS3 and NS5B) and host 18S rRNA gene target to serve as internal control to monitor nucleic acid extraction efficiency and to eliminate potential false negatives. Analytical and diagnostic validation of triplex qRT-PCR assay including in vitro transcribed RNA, synthetic target sequences representing divergent China strains, APPV positive and negative samples from experimental infection studies and/or obtained from other laboratories, and clinical samples submitted to KSVDL demonstrated high sensitivity and specificity of the assay. Phylogenetic analysis of 35 partial NS5B sequences in this study, obtained from clinical samples submitted from different states, indicate high genetic diversity (~83%-100% sequence identity) of APPV within the US. This information also supports the ability of our triplex qRT-PCR assay developed in this study to detect genetic variants of APPV currently being circulated within the US swine herds.

Lalitha Peddireddi, DVM, PhD, Director of Molecular Diagnostic Service, Kansas State Veterinary Diagnostic Laboratory, Kansas State University.  785-532-5661; lalitha@vet.ksu.edu.

Scientific Abstract:

Atypical Porcine Pestivirus (APPV), a highly divergent newly identified pestivirus, is reported as the etiologic agent for type AII congenital tremors in newborn piglets. Since the first report of this virus in the US swine herds in 2015, APPV has been reported in several countries around the world. Sequence analysis suggests high genetic variation (7-17%) among all currently known APPV strains reported from different parts of the world. Therefore, a sensitive and reliable PCR-based diagnostic test is critical for accurate detection of APPV. The main objective of this study is to develop a quantitative real-time RT-PCR (qRT-PCR) assay using all available and newly generated sequence information, for reliable detection of all currently known APPV strains. In this study, we have developed a triplex qRT-PCR assay, using all available sequences from GenBank as well as sequences newly generated from diagnostic samples. The triplex qRT-PCR assay included two APPV target regions (NS5B and NS3), to enhance assay coverage to detect highly divergent China strains reported recently, and host 18S rRNA target to serve as an internal control to monitor and eliminate any false negatives. Individual qRT-PCR assays for each target (NS3, NS5B and 18S rRNA) were optimized separately and then combined into a duplex (NS3+18S and NS5B+18S) and triplex (NS3+ NS5B+18S) assays. Analytical and diagnostic validation of singleplex, duplex and triplex assays were performed using in vitro transcribed RNA, synthetic target sequences representing divergent China strains, APPV-positive and -negative samples from experimental infection studies and clinical samples submitted to KSVDL/ISUVDL. Diagnostic sensitivity of individual APPV assays was ~90% (NS3) and 97% (NS5B), respectively. When these assays are combined into a multiplex format, the diagnostic sensitivity of the multiplex assay increased to 100% with a limit of detection of less than 10 copies of APPV target sequences, which corresponds to a Ct of 37. No cross reactivity was observed for any of the assays with other common swine pathogens indicating high assay specificity. Retrospective screening of 1214 clinical samples, submitted to KSVDL over a period of 2 years, revealed ~15% prevalence of APPV in the US swine herds. APPV has been detected in samples from all age groups pigs, suggesting the possibility of APPV persistence in infected pigs. Of all the specimens tested, oral fluids appear to have higher viral loads (as indicated by low Ct) suggesting oral fluids as a better diagnostic specimen for APPV detection. Phylogenetic analysis of 35 NS5B partial sequences from APPV positive clinical samples, obtained from different states, revealed considerable sequence diversity (85.8% to 100% nucleotide identity) among APPV strains within the US. In addition, two full genome sequences obtained in this study exhibited significant sequence diversity (~12%) compared to the first isolate reported from the US in 2014. Notably, our qRT-PCR assay detected APPV in clinical samples submitted from Canada, which is the first report of APPV in this region. In summary, the triplex qRT-PCR assay developed in this study offers rapid and reliable detection of APPV in the US swine herds. Sequence variation among APPV filed strains reported in this study provides a basis for our understanding of genetic diversity and molecular epidemiology of APPV, currently being circulated within the US swine herds.

Assessment of Slaughter Surveillance Based on Oral Fluids Samples

Project #: 16-175

Principal Investigator: Daniel Linhares

Co-Investigators: Jeff Zimmerman & Marcelo Almeida

Institution: Iowa State University

Industry Summary:

Development of practical, affordable, and effective monitoring and surveillance systems (MOSS) for tracking pathogens in swine populations over time and space is crucial to the future of the industry. Although serum is the traditional surveillance sample, oral fluid specimens are increasingly recognized as a bona fide alternative.

The objective of this study was to determine whether MOSS can be done using oral fluid samples collected in an U. S. abattoir. Porcine reproductive and respiratory syndrome virus (PRRSV) and Senecavirus A (SVA) were used to represent endemic and emerging pathogens, respectively.

A total of 36 lots of pigs (300-450 pigs per lot) were included in the study. On-farm oral fluid (n = 10) and serum (n =10) samples collected within two days of shipment to the abattoir were used to establish the reference PRRSV and SVA status of the study groups. At the abattoir, environmental samples were collected immediately before (n= 32) and after (n = 32) the pigs were placed in lairage. Three veterinary diagnostic laboratories (VDLs) tested the sera and oral fluids for anti-PRRSV antibody (ELISA), PRRSV RNA (rRT-PCR), and SVA RNA (rRT-PCR). Environmental samples (n = 64) were tested for PRRSV RNA and SVA RNA at one VDL.

Oral fluids (n = 3 per lot) were successfully collected from 32 lots (89%) at the lairage. All oral fluids (collected at the farm and abattoir) tested positive for PRRSV antibody at all VDLs. PRRSV positivity frequency on serum samples ranged from 92.4% to 94.6% among VDLs, with an overall agreement of 98% among the laboratories. PRRSV RNA was detected on 2%, 18%, and 18% of sera, farm oral fluids and abattoir oral fluids, respectively. Between-VDLs agreement for rRT-PCR on sera, and oral fluids was 98% and 81%, respectively. For SVA testing, all oral fluids, all farm samples tested negative at all VDLs. However, 70% of oral fluids collected at the abattoir tested positive in at least one VDL. Results demonstrate the need to further investigate the source of SVA RNA.

In summary, anti-PRRSV antibodies, and PRRSV and SVA RNA were successfully detected in abattoir oral fluids from pigs. There was a perfect agreement of PRRSV and SVA ELISA results between locations. There is opportunity to improve the between locations agreement of PRRS and SVA PCR testing. Abattoir surveillance based on oral fluids is an alternative to current practices with tests available or endemic and exotic diseases (including African Swine fever virus, Erysipelothrix rhusiopathiae, Influenza A virus, PCV2, PEDV, Classical swine fever virus, Foot-and-mouth disease virus, and PDCoV), but further studies are needed to better understand how to further improve agreement between abattoir and farm group results.

Detection and Differentiation of PCV3 from PCV2a, PCV2b and the Highly Prevalent PCV2d Mutant Strains

Project #: 16-257

Principal Investigator: Jianfa Bai

Institution: Kansas State University

Industry Summary:

The newly identified porcine circovirus 3 (PCV3) is causing problems in swine similar to that caused by porcine circovirus 2 (PCV2). Yet the PCV3 genome shares little similarity to the PCV2 genome. One objective of this study was to develop a molecular diagnostic assay that can detect and differentiate the majority of the field strains of PCV3 and PCV2. As PCV3 is a new virus, there was limited number of genome sequences available. The other objective was to sequence about 50 PCV3 genomes to study how fast the PCV3 genome is changing, and to use the new sequence information to guide the development, or modification of the detection assay developed in this study. Polymerase-chain reaction (PCR) that is the most used detection technology was used in this study. Analyzing all available genome sequences in the PCR assay design may be the most important first step to ensure the diagnostic coverage of the assay. In this study we have analyzed 1907 available PCV2 full- or near-full genomes, and designed two sets of tests that in combination can detect 98.9% of PCV2 strains including PCV2a, 2b, 2c, 2d and 2e genotypes. This is a significant improvement to several current PCV2 detection assays. The PCV3 assay was designed based on the limited 32 genome sequences available at the time of design. The assay was designed to cover all 32 sequences (100% coverage). However, when more sequences become available, both from the public database and from our home-sequenced ones (n=89), the original design had mismatches to a few strains. To overcome this potential issue, a second set of test was designed and in combination with the first design, they covers all 89 sequences with 100% coverage. An internal control is included in the assay to reduce the false-negative rate. Phylogenetic analysis of the 89 PCV3 full genomes indicated that the largest genetic mutation rate for PCV3 is currently 3.2%. Out of 51 PCV3 genomes we sequenced, 37 were unique genomes, and most of them was grouped into different clusters together with published PCV3 genomes of different locations. As most of our home-sequenced samples were collected from the state of Kansas, our data indicated that the mutations in PCV3 strains do not show an geographic distribution pattern, and they rather mutated randomly in the genome. The 3.2% mutation in the PCV3 genome in just two years indicated that the virus is changing, and continued monitoring the evolution of the virus may be necessary to monitor the emerging strains or genotypes of the virus and to modify molecular detection assays accordingly in order to keep assays up to date. Jianfa Bai, PhD, Director of Molecular Research and Development, Kansas State Veterinary Diagnostic Laboratory, Kansas State University. 785-532-4332; jbai@vet.ksu.edu.

Describing the Cull Sow and Cull Hog Market Networks in the US: A Pilot Project

Project #:

Principal Investigator: Benjamin Blair and James Lowe

Institution: Integrated Food Animal Medicine Systems, Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign

Project Summary:

What is the range of locations of sows that enter a slaughter plant? How many stops along the way do they make? How long do they remain the slaughter channel? Currently there is little data to investigate such questions allowing the industry and regulators to make informed decisions about how to respond to an animal disease outbreak. This project set out to collect data from a harvest plant to see if such information could lead to answers to those questions allowing the industry and animal health officials to better make decisions to prevent and control animal health emergencies.

In this study, data was captured from a single cull harvest plant, over a period of one week during the spring of 2017. We collected Premise ID tags of the culls as they moved through the plant and grouped them by shipping lot. This allowed for the final point of collection to be identified for the purposes of this study. The premise IDs were then cross referenced against a database containing origin information for each unique premise ID to identify the cull’s proposed farm of origin.

In total, we collected premise data on 90.4% of the culls that moved through the harvest plant that week. The animals originated from a total of 297 unique source farms. Sows originated from farms in 21 states and Canada. To determine whether movements to plants derive locally or nationally the distances between origin farms and plant were calculated. We defined the local region for this plant as the radius needed to meet the plant’s capacity at an industry standard 50% cull rate per year. USDA census surveys where used to calculate the breeding inventory of this area at a county level, and determined a 250km radius sufficient to provide the culls to meet capacity. With this in mind, 23.5% of culls originate from farms in the described local region and 43% of final collection points also reside within 250km of the plant. This depicts nature of the cull movements in the market network as national.

The data above presents information on how the cull network begins and ends however little is known about how culls move through collection points. To learn more about how these culls move after leaving the farm and before arriving at the plant, a simple distribution of the distance between the farm of origin and the final collection point was graphed. We also screened the data for statistical outliers and found that culls originating from distances greater that 240km from the terminal collection point were classified as outliers in the network. The majority of culls (86%) originate less than 240km from the final collection point. This interaction is deemed to be a primary interaction meaning that it is very likely the culls moved direct from the farm of origin to the final collection point. 14% of the culls travel a distance greater than 240km to the terminal collection point. Of these 14%, 17.7% or 2.5% of all culls traveled 5 times as far to the last point of collection from the farm than they did from collection point to plant. We hypothesize that 2.5% to 14% of culls moved between multiple collection points prior to arrival at the harvest plant.

We believe to be the first data set collected that allows for this level of detail in describing cull movement from farm until harvest. Although or study has limitations in both the size of dataset and limited timeframe, we believe it provides a unique insight into animal movements and serves as a platform for further work such as this, using larger sets of data to be completed. A better understanding of how culls move throughout the network may provide more detail about disease transmission in the cull market in the US.

Identifying 90.4% of culls over a short time period demonstrates that tracking culls through harvest plants is a realistic method to capture the complexity of the cull network. Although only 2.5%-14% of culls are believed to have moved between multiple collection points prior to harvest. We believe that this is significant and suggest, as was suspected for PEDV, that culls could be an efficient means of transferring diseases across large geographical regions. Being able to understand the way not only sows but diseases move through the slaughter chain holds great value in making the correct decisions to effectively control and prevent disease outbreaks, and why further work must be completed to effectively and efficiently track culls sows through harvest plans to prepare for such an event.

SHIC – Final Research Grant Report

Project #: 15-195

Principal Investigator: Pablo Pineyro, DVM PhD (pablop@iastate.edu) and Luis Gimenez-Lirola, PhD (luisggl@iastate.edu)

Institution: Iowa State University

Industry Summary:

The specific aims of this proposal are to develop a set of diagnostic tools that allows direct detection of SVA.

The first objective was to develop and evaluate a SVA immunofluorescence assay (IFA) for the detection virus in cell culture. The development of this technique has a tremendous impact for confirmation of virus isolation. Once SVA is isolated from clinical samples, direct IFA is the technique of choice to confirm the presence of the virus. Thus, we were able to stain infected cells having a more objective method to confirm infection. In addition, this method, compared to a PCR assay, allows us to identify presence of viable virus. For this specific objective we developed reagents that were not commercially available and now are not only available for ISU Veterinary Diagnostic Laboratory’s diagnostic use, but for researchers and other diagnostic laboratories as well.

Our second objective was to develop a technique that allows identification of the virus in clinical specimens fixed in formalin. Since SVA vesicular lesions are non-specific, this technique is important to detect the virus in lesions and differentiate SVA from other potential causes of vesicular disease. We generated two different antibody reagents that can be used to detect SVA in sections of skin with vesicular lesions. These two antibodies were not commercially available and are now not only available for ISU diagnostic laboratory but for researchers and other diagnostic laboratories.

The third objective was to develop a technique that allows visualization of viral genetic material in clinical specimens. This technique uses fluorescent molecular probes that target two different regions of the virus. In order to reduce the effort and cost involved in fluorescent detection, we further evaluated this probe for detection of SVA with light microscopy. This technique will allow efficient detection of SVA in lesions without the burden of expensive fluorescent scopes. The benefit of molecular detection of SVA in tissues over viral detection by PCR is that can we can also demonstrate viral location in tissues, which will help to understand where and how long the virus can persists in tissues.

In conclusion, we successfully developed a set of reagents that can be used in different diagnostic techniques for virus identification in tissue. These techniques will have a great impact on SVA diagnosis in cases of vesicular disease, providing and supporting the differential diagnosis with other causes of vesicular disease such as foot and mouth disease.

Final Report: Duration of Senecavirus A Shedding From Clinically Affected and Non-affected Sows and Piglets After a Breeding Herd Infection

Project #: 15-206

Principal Investigator: Dr. Chris Rademacher

Institution: Iowa State University

Industry Summary:

This study was designed to evaluate the length of shedding of Senecavirus A (SVA) from a sow farm undergoing an outbreak of SVA in the fall of 2015. Goals were to evaluate the SVA shedding patterns of sows and piglets by PCR and Virus Isolation. In addition, the information obtained regarding SVA shedding pattern should provide some guidance on how long sow herds should be closed to minimize the risk of transmitting the virus to other herds or end point sow cull markets. Tonsil, rectal swabs, and serum were collected from sows and their piglets for 6 consecutive weeks. In sows, PCR results indicated that SVA RNA was detected at low levels out to 6 weeks post outbreak in tonsil and rectal swabs, while detectable levels of SVA RNA in serum were only observed for 3 weeks post outbreak. There was no viable virus isolated from any sow samples. In piglets, PCR results indicated that Senecavirus RNA was detected at low levels (20-40% positive) out to 3 weeks post outbreak in tonsil, rectal swabs, and serum. SVA was isolated in <10% of piglets during weeks 1 and 2 post outbreak, but all were negative by the third week. These findings may suggest that SVA is most likely a short-term risk to other herds and the risk of transmitting Senecavirus A may be lower after 30 days.

Characterization of Seneca Valley Virus Circulating in the US and in Brazil

Project #: 15-192

Principal Investigator: Diego G. Diel1

Co-Investigators: Travis Clement1, Eric Nelson1, Jane Hennings1, Steven Lawson1, Luizinho Caron2, Rejane Schaefer2

Institution: 1South Dakota State University, 2EMBRAPA Swine and Poultry

Industry Summary:

Senecavirus A (SVA) or Seneca Valley virus (SVV) is a picornavirus that was originally identified as a cell culture contaminant in the US in 2002. Subsequent sequencing of unidentified picornaviruses viruses isolated from pigs with a variety of clinical presentations revealed the presence of SVV in the US swine population since 1988. In the past ten years, scattered reports have described the association of SVV with cases of swine idiopathic vesicular disease (SIVD) in New Zealand, Australia, Canada, and the US. Most importantly, since November 2014 there have been increased reports of SVV associated with vesicular disease in swine in Brazil and since July 2015 in the US. The significance of this newly emerging virus lies on its association with vesicular lesions that are indistinguishable from those observed in other high consequence foreign animal diseases (FAD) of swine (i.e foot-and-mouth disease virus, FMDV). Thus, any evidence of vesicular disease in pigs requires a complete diagnostic investigation to rule out the possibility of a FAD. In spite of being present in the US since late 1980’s, there is very limited information on SVA epidemiology. Most importantly, the prevalence of SVV infection and the genetic diversity of viral strains currently circulating in the field remain largely unknown.

Final Report: Development of Reagents and Serological Assays for Seneca Valley Virus

Project #: SHIC #SA1600754; NPB project #15-188 SHIC

Principal Investigator: Steven Lawson

Co-Investigators: E. Nelson, D. Diel, A. Singrey, T. Clement, J. Christopher-Hennings

Institution:South Dakota State University

Industry Summary:

The overall objective of this proposal was to develop and validate diagnostic reagents and tests for Senecavirus A (SVA) antigen and antibody detection. The Specific objectives include:

  1. The development of specific expressed protein and antibody reagents for diagnostic assay development and confirmation of virus isolation attempts, including reagents for immunohistochemistry (IHC), fluorescent antibody (FA) staining and development of serological and antigen capture assays.
  2. The development and validation of first generation serological assays for detection of antibody responses to SVA. These assays included an indirect ELISA, fluorescent microsphere immunoassay (FMIA) and a fluorescent focus neutralization (FFN) assay.

Interim Report: Development of Direct Detection Methods for in situ Diagnostic of Seneca A Virus

Project #: 15-195

Principal Investigator: Pablo Pineyro, DVM PhD and Luis Gimenez-Lirola, PhD

Institution: Iowa State University

Industry Summary:

The specific aims of this proposal are to develop a set of direct diagnostic tools that allows direct detection of SV-A in situ.

Interim Report: Pineyro in situ Diagnostic of Seneca A Virus

Project #: 15-195

Principal Investigator: Pablo Pineyro, DVM PhD and Luis Gimenez-Lirola, PhD

Institution: Iowa State University

Industry Summary:

The specific aims of this proposal are to develop a set of direct diagnostic tools that allows direct detection of SV-A in diagnostic tissues.

A. Development of SVA immunofluorescence assay (IFA) for the detection viral antigen in infected cell culture (Completed).
This objective has been completed on time. The main idea was to develop a tool that allows us to confirm the virus in cell cultures. The development of this technique has a tremendous impact for confirmation of virus isolation. Once SVA is isolated from clinical samples direct IFA is the technique of choice to confirm the presence of the virus. Basically, we will be able to stain infected cell having a more objective way to confirm infection through viral staining with specific antibodies. As compared to a PCR assay, this allows us to identify presence of LIVE virus.
For this specific objective we developed reagents that were not commercially available and now are not only available for ISU Veterinary Diagnostic Laboratory’s diagnostic use, but for researchers and other diagnostic laboratories as well.

B. Development of SVA-IHC for detection of viral antigen in clinical specimens (Currently under development).
This objective has been partially achieved. We proposed to develop a technique that allows us to detect SVA in fixed tissues. Since SVA vesicular lesions are non-specific, this technique will allow us detect the virus in lesions and allows us to differentiate SVA from other potential causes of vesicular disease. In order to achieve this goal, we proposed to generate two different types of antibodies (polyclonal and monoclonal). The development of a polyclonal is complete and already evaluated with excellent results. We are able to detect SVA in section of skin with vesicle. Polyclonal antibodies are easier and faster to produce, but they have less specificity than monoclonal antibodies; thus, they may allow for some cross-reaction and can be more difficult to interpret.

In order to provide a more refined diagnostic tool we also proposed to develop a monoclonal antibody. This technique is still under development. The development of this reagent is done in mice and takes approximately 3 months to complete the process, including multiple steps that cannot be accelerated: the mouse must produce sufficient antibodies to test, and as this is an immune response, takes time to build. Unfortunately, our first attempt provided poor quality antibodies therefore we are repeating the production of new candidate monoclonal antibodies to achieve maximal applicability. We are in the process of screening and evaluating the new SVA-Mab.

For this specific objective we developed reagents that were not commercially available and now are not only available for ISU diagnostic laboratory but for researchers and other diagnostic laboratories.

C. Development of SVA in situ hybridization (fluorescent and/or chromogenic) for direct visualization of viral nucleic acid in clinical specimens (Completed).
This objective has been completed on time. We evaluated a set genetic probes that allows us to confirm the presence of the virus in tissues based on the presence of genetic material. We evaluated two set of fluorescent probes targeting different regions of the virus. One of them (VP1) showed to be adequate to detect SVA genetic material in tissues. In order to reduce the effort and cost involved in fluorescent detection, we further evaluated this probe for detection of SVA detection material with light microscopy. SVA VP1 probe was shown to be efficient detecting viral genetic material with light microcopy. This technique will allow us to detect SVA efficiently in lesions without the burden of expensive fluorescent scopes. The benefit of this over PCR is that it will allow us to detect the exact location of virus, which will help to understand where and how long the virus persists in tissue.

Final Report: SHIC Emerging Disease Fact Sheet

Project #: 15-181

Principal Investigator: James Roth

Objective:

Perform a literature review for each of the disease listed—encephalomyocarditis virus (EMCV), filoviruses: African (e.g., Ebola) and Reston species, Getah virus (GETV), hepatitis E virus (HEV), influenza C (IVC) and D (IVD) viruses, Japanese encephalitis virus (JEV), Menangle virus (MenPV), Nipah virus (NiV), porcine adenovirus (PAdV), porcine astrovirus (PAstV), porcine cytomegalovirus (PCMV), porcine kobuvirus (PKoV), porcine rubulavirus (“blue eye”, PoRV), porcine sapelovirus (PSV), porcine sapovirus (PSaV), porcine teschovirus (PTV), porcine torovirus (ToV), pseudorabies virus (PRV), Sendai virus (SeV), Seneca Valley virus (SVV, also known as Senecavirus A), swine papillomavirus (SPV), swine pox virus (SwPV), vesicular exanthema of swine virus (VESV), and vesicular stomatitis virus (VSV). Develop a literature review and a one-to-two page overview for each of the diseases listed that includes etiology; cleaning and disinfection; epidemiology; transmission; pathogenesis, clinical signs, and postmortem lesions associated with infection in swine; diagnostic tests; immunity; prevention and control; and gaps in preparedness.

Develop a document, including a summary matrix, with information on available diagnostic tests for each of the transboundary production diseases listed in objective, as well as gaps in diagnostic preparedness.

Final Report: Expedited Look Into the Prevalence of Senecavirus A in U.S.

Project #: 15-185

Principal Investigator: Main R, Rossow S, Gauger P, Harmon K, Marthaler D, Vannucci F, Zhang J.

Objective:

Expeditiously obtain some insight to better understanding the prevalence of Senecavirus A (Seneca Valley Virus) currently (8/24/2015 – 9/01/2015) circulating in U.S. swine herds that are not known to be exhibiting clinical signs of acute lameness accompanied by the presence of vesicular lesions on the snout, coronary band, and/or hoof.

Final Report: Evaluation of Disinfectants Against Seneca Valley Virus

Project #: 15-187

Principal Investigator: Goyal, Sagar M.

Objective:

The overall objective is to evaluate the efficacy of certain disinfectants on the inactivation of Seneca Valley Virus (SVV) applied to various surfaces including cured cement, aluminum, stainless steel, and plastic and rubber boots at two different temperatures (40C and ~250C).

Final Report: Seneca Valley Virus Outbreak Investigations

Project #: Systematic epidemiological investigations of cases of Senecavirus A in United States swine breeding herds

Principal Investigator: Holtkamp, Derald, et al.

Objective:

The objectives of this project were to enhance the industry’s knowledge of Senecavirus A’s (SVA) spread and prevention by investigating new cases in a timely, efficient, and uniform manner and to determine the most common gaps in biosecurity that may have led to the introduction of SVA in farms we investigated.

Interim Report: Seneca Valley Virus Genetic Diversity Project Summary

Project #: 15-193

Principal Investigator: Diel, Diego G.

Objective:

To determine the complete genome sequence of SVA strains currently circulating in the United States and in Brazil and to compare SVA complete genome sequences and to identify genetic signatures that might affect the specificity of SVA diagnostic tests.

Final Report: Seneca Valley Virus Shedding Pattern on One Sow Farm in Minnesota

Project #: 15-199

Principal Investigator: Tousignant, Steve, et al.

Objective:

The objectives of this study are to first identify an affected case herd, then conduct an epidemiological investigation and social network analysis, as well as perform longitudinal sample collection on the sow farm to asses shedding patterns of sows, gilt pens and suckling piglets, and develop an archive of samples to be made available for future diagnostic investigations.

Published: Longitudinal Study of Senecavirus A Shedding in Sows and Piglets on a Single United States Farm During an Outbreak of Vesicular Disease

Project #: 15-199

Principal Investigator: Tousignant, Steve, et al.

Conclusion:

The study illustrates the variation of SVA shedding patterns in different sample types over a 9 week period in sows and piglets, and suggests the potential for viral spread between piglets at weaning.