Project #: 21-109 | Principal Investigator: Maria Sol Perez Aguirreburualde | Institution: University of Minnesota
Since 2018, the continuous expansion of ASF through Asia, Europe, and the Americas has kept raising concerns in the industry, resonating with the events of 2013, when a devastating epidemic of porcine epidemic diarrhea (PED) virus caused far-reaching losses. Those events demonstrated the importance of developing systems to provide situational awareness to stakeholders in near-real time to coordinate actions between government agencies and the industry with the ultimate objective of preventing or at least mitigating the impact of disease epidemics. The swine industry is vulnerable to the introduction of pathogens, and their variants, from which the US is currently free. We have developed a private- public-academic partnership to support a system for near real-time identification of hazards that will contribute to the mission of assessing risks to the industry. Identified hazards were shared monthly with swine practitioners and the government to help increase the country's awareness and preparedness. Ultimately, the system has kept contributing to identifying and early detection or creating awareness of key stakeholders to support current prevention and mitigation strategies for the introduction of foreign pathogens into the US.
Project #: 21-109 | Principal Investigator: Dr. Montserrat Torremorell | Institution:University of Minnesota
Among all infectious agents, airborne pathogens are the most difficult to control and in today’s agricultural settings, airborne animal diseases are the most difficult to contain. This is especially relevant for swine reared in confinement and in controlled ventilated environments where exhaust fans help transport particles from inside to outside of the facility. We conducted a review of technologies directed at removing or/and inactivating pathogens for their application to swine farms and found that only air filtration solely consisting of fibrous filters is an established and implemented technology. There are also industrially and medically implementable technologies named electrostatic precipitators and ultraviolet-C systems (both in-duct and upper room), that they are commonly implemented in other industries but are less prevalent in livestock management. Nonetheless, because they have demonstrated scalable performance in aerosol particle removal and/or bioaerosol inactivation, they merit discussion and consideration towards agricultural biosecurity. Lastly, there are emerging technologies, consisting of more recently developed technologies, typically at the laboratory or bench scale that have not been tested at the scales (building sizes and flow rates) required for agricultural biosecurity and are hence several developmental steps away from direct implementation. Lastly, there are protocols and practices ready to be used at the farm level (e.g. wind breaks, covering of fans, movement of infected animals to other sites, depopulation and reduced ventilation) that conceptually, could be employed in the face of a disease outbreak, but their direct impact on reducing the emission airborne pathogens needs to be evaluated and quantified.
Project #: 21-116 | Principal Investigator: John M. Drake | Institution: University of Georgia
A collaboration between SHIC and the Center for the Ecology of Infectious Diseases (CEID) at the University of Georgia examined spillover risk of bacteria from wild North American mammal species into the U.S. swine herd.
The first phase of the project consisted of a data-driven horizon scan which identified 102 bacteria species hosted by 127 North American wild mammal species that have a measurable chance of associating with domestic pigs. These bacteria species were further analyzed and assigned a propensity score based on their predicted ability of spilling over from their wild mammal hosts into domestic swine. A classification of either novel or known was also assigned based on whether or not these species had a known association with pigs documented in academic literature.
The second phase of the project involved a qualitative approach through the use of a survey of subject matter experts (SMEs). The goal of the survey was to rank high-risk bacteria species to assess their potential impact on the swine industry. Five industry professionals completed the survey and identified 16 bacteria species as being of high impact to the swine industry, four of which are not yet known to infect domestic swine: Anaplasma bovis, Clostridium botulinum, Klebsiella pneumoniae, and Yersinia pestis. Additionally, 7 bacteria were ranked as having a high-risk of antimicrobial resistance, and 5 bacteria were ranked as having a high human outbreak potential.
Future steps involve continued efforts to investigate pathways for potential spillover events, such as shared habitats between wildlife and domestic swine, or pathogen overlap. Additionally, different bacterial strains, serovars, and variants of bacteria species of concern must be closely examined to increase predictive power. Bacteria account for a high degree of morbidity and mortality in swine, causing huge losses to the swine industry and zoonotic risks. Domestic pigs often acquire these pathogens (either directly or indirectly) from wild animals, creating the need for assessments such as these to prioritize bacteria species for targeted prevention strategies. Industry professionals across disciplines hold valuable insights crucial to informing risks related to emerging diseases. Their collective knowledge based on years of expertise fills a gap in the absence of a comprehensive database on the pathogenicity of swine-associated bacteria. A lack of published academic literature also fuels the need to engage SMEs to develop a systematic process for identifying bacterial species of concern to the swine industry, and this research represents a summary of such an endeavor.
The benefit of this collaboration to the industry is that bacteriologists, diagnosticians, and other SMEs will have enhanced information needed to prevent, prepare, and respond to emerging diseases and their potential impact on swine health, welfare, and market.
Project #: 21-113 | Principal Investigator: Gustavo Silva, DVM, MS, PhD | Institution: Iowa State University
Decontamination of fomites coming into sow farms using foggers at supply entry rooms is commonly used to mitigate associated risks. Although this practice has been established, recent research has begun to question the effect of this method to inactivate pathogens, especially in complex situations where pathogens may be shielded by organic material or blind spots (Kettelkamp et al., 2019; Leuck et al., 2020).
Objective: The primary objective of this study is to evaluate the efficacy of temperature and time for inactivating porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) on experimentally contaminated surfaces commonly found at supply entry rooms in swine farms.
Materials and methods: The PRRSV MN184, PRRSV 144 L1C variant or PEDV Viruses were used. The surfaces were diamond plate aluminum and cardboard tested at four temperatures (68 ºF, 86 ºF, 104 ºF and 122 ºF) in combination with six holding times (15 minutes, 60 minutes, 6 hours, 12 hours, 24 hours, and 36 hours). Once the surface temperature reached the desired temperature, the coupons were held for the designated holding time. The negative controls remained at room temperature for 36 hours and the positive controls remained at room temperature for 15 minutes. Three replicates for each treatment were performed and each coupon was inoculated with 2 mL of virus or 2 mL of media for negative controls. Virus titration was performed in each sample separately after the holding time. Regression models and Weibull curves were built to assess the impact of temperature and time on virus inactivation.
Discussion: Under the conditions of this study, PRRSV 144 L1C variant was inactivated from aluminum surfaces by heating coupons to 86oF for 12 hours and for cardboard surfaces by heating the coupons to 86oF for 6 hours. Regarding PRRSV MN184, virus inactivation was possible at 86oF after 24 hours on aluminum and at 104oF after 12 hours on cardboard. PEDV inactivation was achieved at 86oF after 6 hours on aluminum and at 86oF after 12 hours on cardboard. Virus was inactivated after 15 minutes and 1 hour at 122oF in aluminum surfaces for PEDV and PRRSV 144 L1C variant, but not for cardboard.
Overall, regardless of the virus tested on this study, to reduce the risk of virus introduction at swine farms through a supply entry room, it is recommended that materials should achieve 860F, and remain at that temperature for 24 hours. Other options, would be to increase the temperate to 104oF or 122oF but decreasing the holding time for 12 hours.
Project #: 20-078 | Principal Investigator: Hiep Vu | Institution: University of Nebraska-Lincoln
African swine fever (ASF) is a devastating viral disease of domestic pigs with mortality rate that can approach 100%. ASF control mainly relies on strict biosecurity that involves movement restriction, quarantine, and compulsive depopulation of affected herds. Rapid and reliable detection of ASFV infected pigs is critical for the control of ASFV. One desirable property of a diagnostic tests is the capacity to detect viral infection, especially during the incubation time, when the infected animals have not displayed any clinical signs. In this study, we evaluated performance of three pen-side tests for ASFV detection: one PCR test for detection of viral genomic DNA and two lateral flow tests for detection of viral antigens.
The first objective was to determine the time from infection to the earliest detection. Ten pigs were experimentally infected with an ASFV strain. Whole blood and oral swab samples were alternatively collected from five pigs every other day post-infection (dpi) and tested with the three pen-side tests. The pen-side PCR test detected infected pigs starting from 2 dpi when using whole blood and 3 dpi when using oral swabs. It consistently detected infection until the end of the study (10dpi). The antigen test detected infected pigs starting from 3 dpi and no longer detected infection at 10 dpi when using whole blood. The antigen test did not work well when tested with oral swabs. Compared with the reference laboratory real-time PCR test, the pen-side PCR test exhibited 97.8% sensitivity and 100% specificity. The antigen had 100 specificity but only 47.8% sensitivity, mainly because it failed to detect infection from samples collected early or late after infection.
The second objective was to evaluate the diagnostic performance of the tests with samples collected from the field. Whole blood and oral swabs were collected from 205 pigs: 34 positives and 171 negatives as determined by the reference laboratory real-time PCR. All pen-side tests had 100% specificity, regardless of the sample types tested. The sensitivity of the pen-side PCR test was 88.2% and 70.4%, respectively when tested with whole blood and oral swab. The sensitivity of the antigen test 50% and 11.11% respectively when tested with whole blood and oral swabs.
In summary, the results of this study show that the PCR pen-side test has better performance than the antigen test as it can detect infected pigs earlier and for a longer duration after infection than the antigen test. Additionally, the pen-side PCR test works with both whole blood and oral swabs while the antigen test work with only whole blood.
Project #: 21-114 | Principal Investigator: Ganwu Li | Co-Investigators: Jianqiang Zhang, Phillip C. Gauger, Eric Burrough, Bailey Arruda | Institution: Iowa State University
Paramyxoviruses that are known to naturally infect swine include porcine rubulavirus, Menangle virus, Nipah virus, and porcine parainfluenza virus. There are reports of less well-characterized paramyxoviruses associated with central nervous and respiratory disease in pigs. However, none of these viruses are classified in the genus Morbillivirus. In the spring of 2020, the Veterinary Diagnostic Laboratory at Iowa State University received twenty-two porcine fetuses (neonatal mortality, stillbirths, mummified fetuses, and fetuses with moderate autolysis) from six litters submitted from Mexico for diagnostic investigation. PCV2, PCV3, PRRSV, PPV1, and Leptospira sp. were not detected by qPCR or RT-qPCR in any litter. Metagenomics sequencing identified a new virus in the genus of Morbillivirus: Porcine Morbillivirus (PoMV). Other currently known members in the genus Morbillivirus, including measles virus (MeV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), canine distemper virus (CDV), phocine distemper virus (PDV), cetacean morbillivirus (CMV), and feline morbillivirus (FeMV), are highly contagious pathogens and can cause serious human and animal diseases. Therefore, there is an urgent need to determine if this new virus associated with swine fetal death is present and, if yes, its prevalence in the U.S. swine population. A total of 450 clinical samples (brains, lungs, and spleens from neonatal mortalities, stillbirths, and mummified fetuses) were collected from all over the United States and subjected to PoMV rRT-PCR assay. Our current assessment has not found PoMV in the U.S. swine populations tested thus far. In addition, virus isolation would be valuable for future characterization of PoMV (diagnostics, prevention, pathogenicity, and pathogenesis investigations). Isolation of PoMV in various cell lines (MARC-145 (a clone of the MA-104 cell line, ATCC CRL-2378), MDCK (ATCC NBL-2), PK-15 (ATCC CCL-33), ST (CRL-1746), Vero (ATCC CCL-81), BHK-21 (CCL-10), LLC-MK2 (CCL-7), ZMAC (ATCC PTA-8764), and primary porcine kidney cells using available PoMV PCR-positive tissue homogenates was attempted in this study. Unfortunately, a PoMV isolate that could efficiently replicate in cell culture has not yet been obtained.
Project #: 20-073 | Principal Investigator: Derald Holtkamp, DVM, MS | Co-Investigators: Daniel Linhares, PhD, DVM, MBA; Kate Dion, DVM | Institution: Iowa State University College of Veterinary Medicine
The swine industry has committed considerable effort to researching and improving biosecurity practices in swine breeding herds; however, attention to biosecurity in the wean-to-market phase of production lags. Events or characteristics of premises that are more frequently associated with introducing PRRSV and porcine enteric coronaviruses in wean-to-market premises are not well understood compared to the swine breeding herds.
The objective of this study was to detect the introduction of wild-type PEDV, PDCoV, TGEV, and PRRSV into groups of growing pigs and to associate the timing of the introductions with the frequency and timing of pathogen-carrying agent entry events.
Seventy-five groups of growing pigs were enrolled in the study originating from sow farms that were consistently weaning pigs that were negative for PRRSV (AASV Category II-vx or IV) and coronaviruses, including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV). Eight pen-level oral fluid (OF) samples were collected from each group of pigs at placement and every two weeks until the end of the wean-to-market phase for all the groups enrolled in the study and tested via PCR for PRRSV, PEDV, PDCoV, and TGEV. Pathogen-carrying agent entry events and biosecurity characteristics for each group were tracked on a two-week basis for each group of pigs paired with the diagnostic results. The categories included premises demographics, people movement, vehicles/deliveries, carcass disposal, air and water entry, swine movements, manure removal, entry of other animals, and disease status.
Overall, 15.5% of groups became positive for PEDV, 26.4 % became positive for PDCoV, and 97.3% became positive for PRRSV. No cases of TGEV were detected.
During the nursery phase, groups that tested PEDV positive had a higher number of average wean pig delivery events, feed deliveries, propane and fuel deliveries, new supply entries, caretaker visits, and maintenance performed outside the barns than nursery premises that tested negative for PEDV. PDCoV positive nursery groups had a higher average number of wean pig delivery events, feed deliveries, propane and fuel deliveries, new supply entries, transferred supply entries, caretaker visits, maintenance performed inside the barns, maintenance performed outside the barns, visits by management, veterinarians, and other visitors compared to nursery groups that tested negative. Entry of new supplies and maintenance performed inside the barns were more frequent for nursery groups that tested positive for PRRSV than negative groups.
PEDV positive finisher groups had a higher average number of feeder pig delivery events on clean trailers, market pig removals, feed deliveries, propane and fuel deliveries, caretaker visits, maintenance performed outside the barns, and visits by management, veterinarians, and other visitors compared to PEDV negative finisher groups. PDCoV positive finisher groups had a higher average number of feeder pig deliveries, market pig removals, feed deliveries, propane and fuel deliveries, new supply entry, and caretaker visits compared to PDCoV negative finisher groups. PRRSV positive finisher groups had a higher average number of feeder pig deliveries, market pig removals, cull pig removals, feed deliveries, rendering removal, propane and fuel deliveries, transferred supply entry, caretaker visits, and visits by management, veterinarians, and other visitors compared to PRRSV negative finisher groups.
Project #: 21-109 | Principal Investigator: Montse Torremorell | Institution: University of Minnesota
The request is to complete the literature review of technologies directed to remove or inactivate airborne pathogens and also to bring together a group of industry leaders to obtain input on strategies/procedures for rapid deployment to contain bioaerosols. The plan is to have these meetings during the months of June-August. So far there has been one meeting with veterinarians attending the Swine Disease Eradication Center meeting in February where they expressed the need to rapidly deploy technologies for biocontainment and interest in subsequent discussions.
Project #: 21-074 | Principal Investigator: Ganwu Li | Co-investigators: Loni Schumacher, Nubia Macedo, Panchan Sitthicharoenchai, Rachel Derscheid, Eric Burrough | Institution: Iowa State University
High mortality events due to Streptococcus equi subspecies zooepidemicus (Streptococcus zooepidemicus) in swine in the United States were firstly reported in Ohio and Tennessee in September and October 2019. One and a half year later (February, 2021), two-year-old adult sows from a production system in Indiana experienced increased sow death loss with 66 deaths in a 2400 sow on production herd within a six-week period. To investigate if the Indiana outbreak isolates were similar /different to those isolates from Ohio and Tennessee outbreaks, whole genome sequencing analysis was performed. Four outbreak isolates from Indiana were genetically distant to those isolates caused high mortality events in Ohio and Tennessee in the spring of 2019, while closely related to an S. zooepidemicus isolate from a horse in Iowa, suggesting that more than one strain of S. zooepidemicus could cause high mortality events in the United States. The genome sequence of the Indiana outbreak isolate was further closed using Nanopore sequencing, comparative genomic analysis was performed, and genomic islands and putative virulence genes were identified. Two genomic islands (GI-3 and GI-13) were identified specifically only in the Indiana outbreak isolates, thus could serve as the biomarker for the diagnosis of this particular strain. In addition, M-like protein gene (szM) and the Fic domain-containing protein gene (bifA) were positive in those Ohio and Tennessee outbreak isolates, but absent from the Indiana outbreak isolates. Identification of biomarkers of bacterial virulence will significantly improve our response to future possible outbreaks caused by S. zooepidemicus.
Project #: 20-178 | Principal Investigator: Cassandra Jones | Institution: Kansas State University
Two feed mills and three breed-to-wean facilities were investigated after being diagnosed with porcine deltacoronavirus (PDCoV) with initial suspicion that feed manufacture and delivery processes were involved in disease transmission. Both feed mills were audited and environmental samples collected in areas that were deemed high risk for virus contamination. All breed-to-wean facilities had PDCoV detected as would be expected, while the only positive samples for enteric coronaviruses associated with feed mills were feed delivery trucks. These results indicate that feed delivery surfaces can help spread virus during an ongoing disease outbreak and must be considered when determining the outbreak origin.
Project #: 20-172 | Principal Investigator: Cesar A Corzo | Institution: University of Minnesota
Objective 1 of 4: Monitor trends in pathogens incidence and prevalence – PRRSv, PEDv, PDCoV, Senecavirus and central nervous system associated viruses continue to be monitored. The 2020-2021 season fortunately ended with the fourth lowest PRRSv breeding herd cumulative incidence (25.8%) during the last 11 years of monitoring. During this year we continued to monitor the emergence and dissemination of a new PRRS variant that caused production losses in the Midwest and changed the seasonality pattern historically observed with PRRS with several associated outbreaks occurring during spring 2020. PEDv continued to be present at a low incidence level as the cumulative incidence remained at 3.5%. Our exploratory data analysis showed that reporting PRRS outbreaks and manure pumping activities are associated as 40% of the breaks occurred within 30 days of this event regardless of type of manure storage.
Project #: 21-115 | Principal Investigator: Scott Dee | Institution: Pipestone Research
Viruses of veterinary significance such as African swine fever virus, foot and mouth disease virus, Pseudorabies virus and Classical swine fever virus are known to survive for extended periods in plant-based feed ingredients imported into North America. To mitigate risk, high risk ingredients, such as oil seed meals, are stored in designated facilities for extended periods under controlled environmental conditions to minimize viral infectivity prior to use. The results from this study suggest that a storage period of 30-days at a temperature of 23.90 C are required to reduce virus infectivity in plant-based feed ingredients such as soybean meal. The outcomes of the study are important, since previous storage periods for feed were based only on mathematical half-life calculations, not controlled studies using live pathogens and representative conditions. We now have for the first time, scientifically sound data based on the use of infectious agents and representative conditions to advise farmers, feed mill operators, federal officials, regulatory and practicing veterinarians, and feed industry leadership on how long and at what temperature to store feed and feed ingredients, to minimize risk. Hopefully, this information will enhance the application and efficacy of Responsible Imports protocols as we collectively work to manage the global risk of feed.
Project #: 20-155 SHIC | Principal Investigator: Chad Paulk | Institution: Kansas State University
Soy-based products are known to pose a viable risk to US swine herds because of their ability to harbor and transmit virus. This project aimed to evaluate soy imports into the US as a whole and from foreign animal disease positive (FAD-positive) countries to determine which products are being imported in the highest quantities and observe potential trends in imports from FAD-positive countries. Import data were accessed through the United States International Trade Commission website (USITC DataWeb) and summarized using R (version 4.0.2, R core team, Vienna, Austria). Twenty-one different Harmonized Tariff Schedule (HTS) codes were queried to determine quantities (metric tonnes, MT) and breakdown of different soy product types being imported into the US from 2015 to 2020. A total of 78 different countries exported soy products to the US in 2019 and 2020 with top contributors being Canada (546,467 MT and 481,497 MT, respectively), India (397,858 MT and 430,621 MT, respectively), and Argentina (122,116 MT and 79,471 MT, respectively). Soy oilcake (582,273 MT) was imported in the largest quantities, followed by organic soybeans (270,194 MT) and soy oil (134,436 MT) for 2020. Of the 78 countries, 46 had cases of FAD reported through the World Organization for Animal Health (OIE) World Animal Health Information Database (WAHIS). Top exporters of soy products to the US from FAD-positive countries in 2019 and 2020 were India (397,858 MT and 430,621 MT, respectively), Argentina (122,116 MT in 2019), and Ukraine (40,293 MT and 56,392 MT, respectively). The risk of FAD introduction to the US through soy imports can fluctuate based on where FAD outbreaks are occurring, shipping methods, and end usage of products. A system to monitor these factors could help make future decisions about trade and risk of FAD introduction to US swine herds. Based on information generated from this project the following frequently asked questions (FAQ) and best practices for importation of soy products were compiled below.
Investigators: Fangfeng Yuan1,2, Xingyu Yan1,2, Brandi Feehan2, Rui Guo2, Yanhua Li2, Giselle Cino2, Ying Fang1, 2*, Douglas Marthaler2*
Institution: 1University of Illinois, 2Kansas State University
An emerging porcine sapelovirus was isolated in a diagnostic specimen from a US swine farm, designated as PSV KS18-01. Full-length genome sequence was obtained through next-generation sequencing. Phylogenetic analysis showed that the virus is more closely related to two Japanese strains but is distantly related to two known US strains. PSV specific diagnostic tools were developed, including the monoclonal antibodies again VP1 and VP2, and a VP1-VP2 antigen-based indirect ELISA. Using this assay, the dynamic response of PSV antibody was investigated in a group of post-weaned pigs that naturally exposed with PSV. The availability of the PSV isolate (KS18-01) and the specific diagnostic reagents and assays provide important tools for PSV control and prevention.
Project #: 20-081 | Investigators: Luis G. Giménez-Lirola, Neeraja Venkateswaran | Institution:Innoceleris LLC. and Tetracore Inc.
In the absence of effective African swine fever virus (ASFV) vaccines, infection prevention and control through diagnostic testing and quarantine is critical. Early detection and differential diagnosis of ASFV infections increase the chances for successful control of this devastating disease. However, the interpretation of the ASF diagnostic results can be complicated due to the complex epidemiology of the disease, and its unspecific and highly variable clinical presentation, i.e., same strain producing a wide range of clinical forms. The objective of this proposal was to evaluate the performance of ASFV serum/oral fluid indirect ELISA (iELISA) (collaborative work between Innoceleris LLC. and Tetracore Inc.) for surveillance and monitoring of ASFV outbreaks in commercial farms in Vietnam. For this cross-sectional field study, our field team at Hanoi University collected a 398 paired serum/oral fluid samples, individually collected from each animal, which included 100 samples from 34 ASF-acute farms, 98 samples from 47 ASF-chronic farms, and 200 samples from 20 ASF-negative farms. The samples were tested by Tetracore ASFV iELISA and real-time PCR (qPCR). As expected, the detection rate by qPCR (74% serum; 69% oral fluid) was higher than by ELISA (16% serum; 11% oral fluid) in acute farms due to that most of the animals did not seroconvert yet. Contrary, in chronically affected farms, the detection rate of the ELISA was higher (72% serum; 57% oral fluid) than the qPCR (56% serum; 34% oral fluid). However, when we combined both qPCR and ELISA, the detection rate of ASFV positive animal increased in acute (75% serum; 74% oral fluid) and particularly in chronic farms (85% serum; 74% oral fluid). All serum samples from negative farms were negative by both ELISA and qPCR (100% diagnostic specificity) while, for oral fluids, we obtained 100% and 99% diagnostic specificity for qPCR and ELISA, respectively. The high diagnostic specificity of the tests is particularly important for ASF surveillance. Absence of false positives avoid false alarms and disruption in production, and lack of confidence in the tests/surveillance system. This unprecedented study show that there are no single best diagnostic approach for ASFV surveillance and demonstrate that the combined use of the Tetracore qPCR and indirect ELISA tests and serum/oral fluid sampling, increase efficiency of ASF disease surveillance.
Project #: 20-077 | Investigator: Marie Culhane | Institution: University of Minnesota
An ASF outbreak in the United States (US) will have a significant impact on the swine and allied industries. This proactive semi-qualitative Risk Assessment (RA) was conducted to evaluate the risk movement of liquid-cooled boar semen (semen) during an African Swine Fever (ASF) outbreak in the US swine industry will result in the spread of ASF virus (ASFv) to other premises with swine. Risks of ASFv spread associated with the movement of semen originating from a Monitored Premises within, into, and outside a Control Area were evaluated as part of a public-private partnership between the US swine industry, the College of Veterinary Medicine at the University of Minnesota (UMN), and the Swine Health Information Center. When completed, reviewed, and cleared, the RA can be used to support permits for the safe and timely movement of fresh boar semen during an ASF outbreak. This assessment is applicable to US swine production and considers the implementation of standard boar stud and semen production practices, enhanced biosecurity recommendations (EBRs) proposed by the Secure Pork Supply (SPS) plan (December 2017), and industry-agreed upon targeted mitigation measures determined by the Boar Stud Working Group (WG).
This RA is limited in scope and intended to identify ASFv transmission pathways associated with semen movement and assess the corresponding likelihoods of carrying ASFv from an infected boar stud premises and causing an infection on off-site sow or gilt breeding facilities during an ASF outbreak in the US, despite the implementation of all standard preventive measures as well as outbreak-specific targeted and/or enhanced measures. This RA will ultimately provide the framework necessary for decision-makers to both quickly assess the effectiveness of preventive measures as they pertain specifically to semen movement and also implement a permit system to allow boar studs with no known infection to move semen into, within, and outside of the ASF outbreak Control Area.
The risk evaluation is based on the likelihood of the presence of infected or contaminated semen products. The likelihoods evaluated include 1. A boar stud facility in a Control Area becoming infected with ASFv; 2. Detecting infected boars on the origin premises prior to semen movement; 3. Semen products becoming contaminated prior to movement off the boar stud; 4. Semen products becoming contaminated in transit by fomites; and 5. ASFv release during transportation of infected or contaminated semen. The likelihood that other susceptible pigs can be exposed to ASFv and become infected as a result of insemination or shipment will be evaluated in cooperation with a swine breeding WG as the proactive RA continues.
The overall findings, which require further evaluation, review, and clearance at the conclusion of the RA process, are that the risk that movement of liquid, cooled boar semen from a boar stud premises that qualifies as a Monitored Premises inside a Control Area to an off-site sow gestation or farrowing facility within or outside of a Control Area during an ASF outbreak results in the infection of susceptible swine at the destination premises is likely to be low to moderate if boar studs follow the Enhanced Biosecurity Recommendations [(EBRs) - as outlined in the Secure Pork Supply (SPS) Plan] and as agreed upon by the WG. In addition, as tactical, targeted biosecurity measures are developed with WG consultation, additional mitigations, meant to be targeted EBRs, and, assuming that these additional, strategic, targeted EBRs will be implemented as mitigation measures, the risk will likely be decreased.
This work is an evolving product-specific risk assessment that will be reviewed and updated as necessary before and during an outbreak to incorporate the latest scientific information, preventive measures, and analytical methodology. Updates and revisions to risk assessments are advisable and necessary as new data become available. The opportunity to update risk assessments to address the limitations of previous analytical methods and data might have effects on the risk ratings and outcomes. If the Incident Command System is activated in response to an ASF outbreak, APHIS (and Incident Command staff) will review this risk assessment with respect to the situation in order to assess industry requests for the movement of boar semen.
Principal Investigators: Encheng Sun, et. al. | Institution: Harbin Veterinary Research Institute
The Georgia-07-like genotype II African swine fever virus (ASFV) with high virulence has been prevalent in China since 2018. Here, we report that genotype I ASFVs have now also emerged in China. Two non-hemadsorbing genotype I ASFVs, HeN/ZZ-P1/21 and SD/DY-I/21, were isolated from pig farms in Henan and Shandong province, respectively. Phylogenetic analysis of the whole genome sequences suggested that both isolates share high similarity with NH/P68 and OURT88/3, two genotype I ASFVs isolated in Portugal in the last century. Animal challenge testing revealed that SD/DY-I/21 shows low virulence and efficient transmissibility in pigs, and causes mild onset of infection and chronic disease. SD/DY-I/21 was found to cause necrotic skin lesions and joint swelling. The emergence of genotype I ASFVs will present more problems and challenges for the control and prevention of African swine fever in China.
Project #: 20-103 | Principal Investigators: Giovani Trevisan, Edison Magalhaes, and Daniel Linhares | Institution:Iowa State University
In 2017 SHIC funded the Domestic Swine Disease Surveillance project, currently housed under the Swine Disease Reporting System (SDRS) initiative. The purpose of this proposal was to keep the Domestic Swine Disease Surveillance program ongoing and further develop a capability to improve sustainability over time. The specific aims included a) keep updated the aggregated database with diagnostic data from the participating laboratories, and provide monthly PDF reports to SHIC as well as keep updated the online interactive dashboards; b) enhance the sustainability of the project by migrating the report-building mechanism to an automated R Markdown technology; c) improve data exchange capability between the Data warehouse project, the Domestic Swine Disease Surveillance program, and other SDRS programs by migrating the data visualization platform to Tableau.
During the time of this project, the database was consistently updated with data from participant laboratories. The database was continuously updated and now contains data from more than 950,000 distinct submissions tested by PCR for the five U.S. porcine endemic agents, i.e., porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea (PEDV), porcine deltacoronavirus (PDCoV), transmissible gastroenteritis (TGEV), and Mycoplasma hyopneumoniae. Interactive online dashboards with filtering capabilities, e.g., age category, specimen, geographic region, were kept alive, updated, and are available on the project website (https://www.fieldepi.org/sdrs). Monthly PDF reports have been consistently produced and releases every first Tuesday of the month. Monthly PDF reports are published at the project webpage, SHIC website (https://www.swinehealth.org/domestic-disease-surveillance-reports/), and distributed by email to more than 270 registered receivers from 129 organizations/institutions from 7 countries (US, Canada, Mexico, Brazil, Chile, Germany, and Spain).
In July 2020 (SDRS report # 29), monthly PDF reports started to be produced using the R Markdown technology and gained a new face. An R Markdown script has written and allowed the generation of reports in a standard and consistent format. The R Markdown script was codified to automate as much as possible the process of generating and update images, formatting, and description for the monthly detection changes under each agent page. As an ongoing real-time project, there is still the need for staff to interpret the finding, debugging, communicate results with the advisory group, gather feedback, and compile the final report, including the input and feedback from the Advisory group. The usage of an R Markdown script has enhanced SDRS sustainability by reducing the time needed and errors in compiling the final monthly PDF final report.
Interconnection between databases and different visualization and analytical tools are key elements for project sustainability. The need to efficiently process and interconnect the database with the R Markdown and visualization platforms like Power BI and Tableau requested a redesign of the SDRS database structure. The SDRS has been built using a building block approach. Initially, SDRS started reporting PRRSV detection from Iowa and Minnesota VDLs and later on added South Dakota and Kansas VDLs and additional data for the enteric coronavirus and M. hyopneumoniae. Initially, each agent's database was kept separated. A restructuring was done to compile, combine, and store all data into a single database. This redesign allowed the database interconnection with the R Markdown to generate PDF monthly reports, analytical tools like R and SAS, and business intelligence tools, like Power BI and Tableau, for data visualization.
Additionally to the proposed scope of this project, the SDRS team has consistently produced monthly audio and video reports distributed on the project website, Youtube Channel, Swine Cast platform, and LinkedIn. Also, during the last year, SDRS has been actively engaged in providing information and support to the U.S. swine industry when it went into a crisis due to the activity of contemporary and emergence of a new PRRSV strain threatening the U.S. swine health. Early detection is a key component for containing the spread of emerging or re-emerging animal health threats. More than 110 monitoring algorithms are currently implemented in the SDRS background to detect early changes in the pattern of agent detection.
This collaborative project funded by SHIC has real-time capabilities for data collection, analysis, and sharing of swine health data and information to protect and enhance the health status of the United States swine herd. SDRS is a real-time coordinated and largely representative national swine disease monitoring program that provides VDL information in efforts to minimize disease threats current and future impact. The SDRS is the only publicly available source of swine health information from U.S. VDLs. It is also the only source of information on pathogen activity in all age groups (from boar studs to grow-finish pigs). The sharing of information on endemic and endemic re-emerging diseases affecting the swine population in the U.S. has and continues to assist veterinarians and producers in making informed decisions on disease prevention, detection, and management.
Project #: 21-065 | Principal Investigator: Diego G. Diel | Institution: Cornell University
Feed biosecurity has been an area of significant interest to the swine industry. Early studies with porcine epidemic diarrhea virus (PEDV) suggested that the virus may have been transmitted through feed. Recent experimental evidence confirmed that PEDV, SVA and FMDV can indeed be transmitted through contaminated feed that is ingested naturally by susceptible pigs. One way of reducing the risk of pathogen transmission through feed is to test feed ingredients and feed before they are introduced into farms and fed to pigs. This would only be possible if sampling and nucleic acid extraction methods would allow efficient detection of pathogens in feed. In this study we focused on comparing the performance of three commercially available nucleic acid extraction kits (CORE, IndiMag, MVP II). These kits were tested in samples that were spiked with PRRSV, SVA and PEDV and that were previously collected as part of a transportation study and tested in another VDL. Our results show that the Core extraction kit outperformed the other two kits evaluated in the present study and previously used in another VDL that originally had tested the samples. Overall samples extracted with the Core kit presented lower Ct value (at least for PRRSV and SVA) and a higher sensitivity when compared to samples extracted with MVPII or the IndiMag, One of the key issues that remains to be addressed in future studies is the sampling method to be used for large volumes of feed or feed ingredients.
Project #'s: 18-137 and 18-211 | Investigators: Leonardo C. Caserta1,2, Jessica C. G. Noll1,2,Aaron Singrey1,Megan C. Niederwerder3,Scott Dee4,Eric A. Nelson1,Diego G. Diel1,2 | Institution: 1South Dakota State University, 2Cornell University, 3Kansas State University, 4Pipestone
Animal feed and feed ingredients have recently been investigated as sources of pathogen introduction to farms and as a potential source of infection to animals postconsumption of contaminated feed. Survival of several viruses for a prolonged period has been demonstrated in feed. Here, we determined the rate of decay of Senecavirus A (SVA) in swine feed ingredients as a function of time and temperature and established half-life estimates for the virus. Select feed ingredients were spiked with a constant amount of SVA (105 median tissue culture infectious dose 50) and incubated at 4, 15 and 30◦C for up to 91 days. Virus viability and the presence of viral RNA were assessed in samples collected over time. At the three different temperatures investigated, dried distillers’ grains with solubles (DDGS) and soybean meal (SBM) provided the most stable matrices for SVA, resulting in half-lives of 25.6 and 9.8 days, respectively. At 30◦C, SVA was completely inactivated in all feed ingredients and in the control sample, which did not contain a feed matrix. Although virus infectivity was lost, viral RNA remained stable and at consistent levels throughout the experimental period. Additionally, the ability of SVA to infect swine via ingestion of contaminated feed was investigated in 3-week-old, weaned pigs. Animals were provided complete feed spiked with three concentrations of SVA (105, 106 and 107 per 200 g of feed) and allowed to naturally consume the contaminated feed. This procedure was repeated for three consecutive days. Infection of pigs through consumption of contaminated feed was confirmed by virus neutralization assay and the detection of SVA in serum, feces and in the tonsil of exposed animals by real-time reverse transcriptase PCR. Our findings demonstrate that feed matrices are able to extend the survival of SVA, protecting the virus from decay. Additionally, we demonstrated that consumption of contaminated feed can lead to productive SVA infection.
Project #: 18-194 | Investigators: Carolina Stenfeldt1,2, Miranda Bertram1, Haillie Meek1,3, Ethan Hartwig1, George Smoliga1, Megan Niederwerder2, Diego Diel4, Scott Dee5, Jonathan Arzt1 | Institution: 1Plum Island Animal Disease Center, 2Kansas State University, 3Oak Ridge Institute for Science and Education, 4Cornell University, 5Pipestone
Important work examining the role of contaminated feed as a vector for transmission of foot-and-mouth disease virus (FMDV)was published in July 2021. Specifically, the study performed by researchers from ARS/USDA at Plum Island, evaluated the potential risk of incursion of FMDV into naïve pig herds through contamination of feed. Per the study report, the goal of the project was the assessment of the infectiousness (viability) of FMDV in commercial whole pig feed and pig feed ingredients. Additionally, the researchers, led by Drs. Stenfeldt and Arzt, determined the dose required to infect pigs through natural feeding behavior. Finally, the project looked at the ability of select commercially available feed additives to reduce infectivity of contaminated feed. “While comparable research investigating the potential biosecurity risks of imported feed exists for other viral pig pathogens (Dee et al., 2018; Niederwerder et al., 2020; Niederwerder et al., 2019), this is the first comprehensive evaluation of the risk of FMDV infection of pigs through ingestion of contaminated feed under controlled experimental conditions,” wrote the authors.
Project #: 18-142 | Principal Investigator: Christa Goodell1 | Co-Investigators: Erik Schacht1, Vu Tran-Hoang1, Huy Nguyen1, Quy Tran1, Carlo Maala1, Danh Lai2, Che Thanh Nguyen2, Hien Le2, Nam Nguyen2, Duy Do2, Toan Nguyen2, Minh Nam Nguyen3, Jeffrey Zimmerman4, Oliver Gomez-Duran1 | Institutions: 1Boehringer Ingelheim Vetmedica GmbH, Ingelheim, Germany; 2Animal Sciences and Veterinary Medicine, Nong Lam University, HCM City, Vietnam; 3Research Center for Genetics and Reproductive Health, School of Medicine, Vietnam National University, HCM City, Vietnam; 4Iowa State University, Ames Iowa
Vietnam has lost ~6 million pigs to ASFV since the first reported outbreak in February 2019. The national swine herd has returned to 88.7% of its pre-ASFV level, but the risk of ASFV recurrence is high because of new and on-going ASFV outbreaks (Report #VM2021-0042, USDA & GAIN, May 11, 2021).
Because of the increased value of pork in Vietnam due to reduced supply, standard ASFV control measures have centered around a modified “test-and-remove” or “Tooth Extraction” protocol. A common “Tooth Extraction” protocol for a sow farm is to remove any sow exhibiting clinical signs compatible with ASF (taking a whole blood sample for ASFV PCR testing) plus the two sows in stalls on each side of the index (clinical) animal (Yaros, Leman, 2019).
The objective of this study was to test the efficacy of the “Tooth Extraction” protocol.
For each "ASFV event" (identified by farm caregiver), whole blood samples were collected from the index sow plus 14 animals in gestation stalls on each side of the index sow (see Figure 1). Samples were tested for ASFV DNA by real-time ASFV PCR within 24-hours of arrival to the laboratory. The proportion of positive animals was analyzed as a function of the gestation stall distance from the index sow.
Figure 1. Blood sampling protocol around a suspected ASF clinical sow in gestation†‡
†F0=index sow, F1= direct/closest contact neighbors, F2= indirect contact neighbors; A= ”down” row, B= “up” row
‡(The rationale for the sampling distribution was due to an assumption drinking water flowed down a common water trough)
Results. 766 whole blood samples from 52 ASFV events were collected and tested for ASFV DNA by PCR. 85 samples were positive for ASFV DNA by PCR.
• In 17 (33%) of the 52 events, the index sow and 14 neighbor sows were ASFV PCR negative.
• In 35 (67%) of the 52 events, the index sow was ASFV PCR positive.
- Among allsows detected as ASFV PCR positive, 39 (78%) were located outside of the index animal and her direct contact neighbors (F0, F1A1, F1B1). Thus outside of the index animal and her contact neighbors (F0, F1A1, F1B1), 10% of all animals tested were ASFV PCR positive, compared to the direct contact animals alone (F1A1, F1B1), representing 18% of the all animals tested.
- If the index sow and 4 direct/closest contact neighbors were removed (F0, F1A1, F1B1, F2A1, F2B1), there was a 50% probability that additional ASFV PCR positive (but unidentified) sows remained.
• ASFV DNA was detected in blood from sows showing no clinical signs.
The results of this study suggest “Tooth Extraction” is not sufficient to eliminate ASF from a pig farm.
Project #: 20-175
Discovered in 2001, porcine parvovirus 2 (PPV2) is prevalent in swine in countries worldwide. While it is commonly detected in swine samples, the clinical significance of infection is unclear. We recently identified PPV2 present at high levels in the lung of a pig with pneumonia, prompting this investigation into the role of PPV2 in respiratory disease. Here we report that PPV2 was detected in 39% of lungs submitted for routine diagnostic testing. There was a significant positive correlation between PPV2 viral load and pig age. Histologically, PPV2 was mainly detected in alveolar macrophages in 28% of lungs with interstitial pneumonia, with PPV2 viral load tending to correlate with the number of macrophages in the lungs. While co-infections with PPV2 and established swine respiratory pathogens influenza A virus and porcine reproductive and respiratory syndrome virus were commonly detected, assessment of viral loading and tissue locations showed no obvious association between PPV2 and other viral pathogens. Relatedly, in a third of PPV2 positive lungs analyzed by metagenomic sequencing, no other pathogens were identified. Together, these results suggest that PPV2 may play a primary role in porcine respiratory disease.
Project #: 19-154 | Principal Investigator: Daniel C. L. Linhares, DVM, MBA, PhD | Institution: Iowa State University
PRRS continues to plague the swine industry with 20-30% of herds breaking annually. These outbreaks cost the swine industry and its producers $664 million every year. Swine producers continue to offer efforts to increase biosecurity with the intention to provide the best welfare for their pigs and produce a wholesome product for consumers. The industry needs the ability to better predict risk of a PRRS outbreak as well as to evaluate the level of biosecurity in their farms. The objective of this study was to measure and benchmark the relationship between key biosecurity aspects and PRRS outbreaks in breeding herds, while validating a short biosecurity screening survey (44 questions). The survey captured data on herd demographics, PRRS outbreak history, frequency of high-risk events, surrounding swine density, transport biosecurity practices, carcass disposal, and people movement. Three machine-learning algorithms were used to identify key biosecurity factors and practices associated with PRRS outbreaks. The most accurate, based on ability to explain variability between farms in the frequency of reported PRRS outbreaks, was selected. Moreover, positive predictive value (PPV) from the models were utilized as a biosecurity score. We investigated the correlation between PPV and the reported frequency of PRRS outbreaks.
Thirteen production systems from 15 states were enrolled in the study (n=188 sow herds). The best machine learning algorithm predicted PRRS occurrences with an accuracy of 78.5%. The 44 biosecurity measures were ranked according to their contribution to the model prediction. The first four most important variables were devotion to breeding genetic replacements, number of swine premises within a three-mile radius, number of breeding females on the premises, and distance to the nearest public road. The probability that a herd had reported an outbreak were higher in farms that had raised breeding animals as genetic replacements. The higher the number of swine facilities within a three-mile radius and the closer the farm was to a public road, the more likely the facility would be expected to break with PRRS. Finally, the PPVs were correlated with the number of reported PRRS outbreaks (correlation of 72%). The majority of farms that had reported no outbreaks in the past five years had low (<50%) probability of having a PRRS outbreak (i.e., low score) while a larger number of PRRS cases were expected for farm who had reported at least one outbreak (i.e., high score), with few exceptions.
The analysis offers a flexible, shortened approach to screen breeding herd’s PRRS biosecurity vulnerability. This study highlights the value of using data to build upon our understanding of biosecurity risk in an operation.
Project #: 20-071 | Principal Investigator: Hiep Vuo| Institution: University of Nebraska-Lincoln
African swine fever virus (ASFV) is the most devastating viral pathogen which can cause up to 100% mortality in domestic pigs. Currently, the virus is affecting many swine producing countries, leading to the approximately 25% reduction of global swine population. Swine transportation plays a major role in spreading infectious pathogens. Thus, developing a procedure for effectively disinfecting animal trailers will help reduce viral spreading. The most effective method to effectively inactivate pathogens in transportation trails is a combination of washing, disinfecting and drying. However, there are not enough washing facilities to wash all trailers between loads of swine. Therefore, we are interested in testing if it is possible to effectively inactivate ASFV in the presence of organic materials (feces, bedding) through the use of thermal-assisted drying and decontamination (TADD) which commonly operates at the temperature between 63°C and 71°C (Dee et al., 2005).
The objective of this project is to determine the optimal baking time and temperature required to completely inactivate ASFV in aluminum surface contaminated swine feces. We tested the inactivation efficiency of contaminated trays under 3 conditions:
• Baking contaminated aluminum trays at 54oC and 63oC for 5, 10, and 15 min
• Power washing the tray surface with water at room temperature followed by baking at 54oC and 63oC for 5, 10, and 15 min
• Power washing the tray surface with water, spray disinfectant (Virkon-S) followed by baking at 54oC and 63oC for 5, 10, and 15 min
Two different methods were used to evaluate the efficiency of the treatments: PCR to detect of viral genomic DNA and virus isolation to detect infectious virus.
In the first condition, swabs collected from contaminated trays at all time-points post incubation at 54oC and 63oC were positive by PCR, indicating that heat treatment could not eliminate viral genomic DNA. On the other hand, swabs collected from contaminated tray at 5 min post incubation at either 54oC or 63oC were negative by virus isolation, indicate that holding ASFV in the presence of feces at 54oC for 5 min is sufficient to inactivate the virus.
In the second and third conditions, only two swabs collected after washing were positive by PCR at high Ct value (e.g. 37.03). These swabs were negative by for virus isolation. Thus, under the conditions of this study, power washing of the trays with water at room temperature was efficiently remove contaminated material from the trays.
Collectively, results obtained from this research provide valuate information for the develop effective sanitation protocols to disinfect animal trailers to reduce the spreading of ASFV.
Project #: 19-236 | Principal Investigator: Nubia Macedo | Institution:Iowa State University
In swine, Streptococcus equi subsp. zooepidemicus (SPZ) caused a severe outbreak in China in 1975. In the United States, a genetically similar and hypervirulent SPZ strain caused high mortality outbreaks in sows and feeder pigs in 2019. There is currently no available challenge model in pigs to evaluate the disease process and diagnostics, which would provide meaningful information about the pathogenicity and epidemiology of SPZ in pigs. To address this need, conventional 6-week-old pigs were challenged using hypervirulent SPZ strains and a genetically different, less virulent SPZ strain. Additionally, sequenced SPZ strains were assessed to identify specific virulence markers, and a multiplex RT-PCR assay was designed for SPZ identification and prediction of virulence in swine isolates. Pigs challenged with the hypervirulent SPZ strains developed severe systemic disease, with mortality varying from 50-100%. In contrast, pigs challenged with the less virulent swine strain had minimal clinical signs and no mortality. The colonization and systemic distribution of SPZ were evident in challenged pigs by culture and PCR. This is the first study to experimentally infect and reproduce the disease in weaned pigs with a hypervirulent swine SPZ strain. Furthermore, pathogenicity differences between genetically different swine strains were described. This experimental model will allow for further investigations on the pathogenesis of SPZ in swine and the development of methods of prevention and control of this emerging disease. The newly developed multiplex RT-PCR provides an accurate and timely assay for detecting and monitoring SPZ in swine herds.
Project #: 19-235 | Principal Investigator: Cesar A Corz | Institution: University of Minnesota
Objective 1: Monitor trends in pathogens incidence and prevalence – PRRSv, PEDv, PDCoV, Senecavirus and central nervous system associated viruses continue to be monitored. The 2019-2020 season fortunately ended with the third lowest PRRSv breeding herd cumulative incidence (24.5%) during the last 11 years of monitoring. During this year we saw a different trend in breeding herd PRRSv prevalence as the proportion of herd staying in category 1 increased and “plateaued” which had not been seen before. PEDv and PDCoV continued to be present at a low incidence level. Mycoplasma hyopneumoniae was included into our monitoring program through a convenient sample of 8 systems. We now have the ability to quickly add new pathogens, which allow us to be prepared in the case of a FAD introduction.
Objective 2: To conduct prospective monitoring of PRRSv sequence evolution and impact – PRRSv sequence acquisition has been stabilized and these are being acquired on a monthly basis. Throughout the year, several participants contacted us to provide outbreak investigation support. A total of 29 sequence analysis were conducted with one of the latest being a virulent 1-4-4 virus. This allowed us to become a neutral third-party curating sequence to identify similarities and putting systems in contact whenever both parties agree.
Objective 3: Develop capacity to capture and analyze movement data – Transport data is acquired actively and has been analyzed. Movement data can be obtained at a granular level allowing for traceability but most importantly, allows the producer to follow the truck in real-time. Transport biosecurity compliance continues to become an achievable goal including every single step between the truck-wash, loading of pigs, unloading and return to truck-wash. Characterization and description of transport data has shown that few transport vehicles come in contact with 1/3 of the farms of the participating production system highlighting an important level of connectivity.
Objective 4: To expand participation of producers to allow for all to be involved – Expansion continues at three levels: sow, boar and growing pig populations. A new production system joined the SHMP during the year. A total of 30 boar studs from 12 participants have been added to our database. The growing pig population continues to grow with 4 companies sharing their growing pig locations. Work is being done towards linking sow-growing pig populations in our database.
Project #: 19-229 | Principal Investigator: Andres Perez | Institution: University of Minnesota
The expansion of ASF through Asia has raised concerns in the industry, resonating the events of 2013, when a devastating epidemic of porcine epidemic diarrhea (PED) virus caused far-reaching losses. Those events demonstrated the importance of developing systems to provide situational awareness to stakeholders in near-real time, to coordinate actions between government agencies and the industry with the ultimate objective of preventing or at least mitigating the impact of diseases epidemics. The swine industry is vulnerable to the introduction of pathogens, and their variants, from which the US is currently free. We have developed a private- public-academic partnership to support a system for near real time identification of hazards that will contribute to the mission of assessing risks to the industry. Identified hazards were shared monthly with swine practitioners and the government, to help increase the country awareness and preparedness. Ultimately, the system has kept contributing to identify and early detect or create awareness on key stakeholders to support current prevention and mitigation strategies for introduction of foreign pathogens into the US.
Project #: 19-153 | Principal Investigator: Kimberly VanderWaal | Institution: University of Minnesota
Despite our growing understanding of the epidemiology of viral diseases such as Porcine Epidemic Diarrhea virus (PEDv) and Porcine Reproductive and Respiratory Syndrome virus (PRRSv), a gap remains between the science and the ability of producers to be able to effectively estimate and respond to spatial and temporal variation in risk. Therefore, the aim of this project was to generate farm-level forecasts of PEDv risk that account for recent animal movements, present disease distribution, and environmental factors. Utilizing data captured by the Morrison Swine Health Monitoring Project, we built machine learning algorithms that predict whether a sow farm will break with PED two weeks in advance.
Our forecasting tool was able to detect approximately 20% of the outbreaks in a region (sensitivity), while 70% of the outbreaks our tool said would occur did indeed happen (positive predictive value).The most important factors related to breaks were animal movements both into the sow farm and its neighbors, as well as the PED-status of the origin farm of these movements. Farm density and ambient temperature were also important predictors. We developed this tool using data for a single US swine-dense region and began applying it to a second region of the country, operating with different industry systems and a different environment. This forecasting tool is operationalized for real-time use – since December 2019, partnering systems have received weekly system-specific predictions of PEDv outbreak risk at farm level. These predictions are made two weeks into the future, which allows systems, veterinarians and producers to act in case a high probability of an outbreak is forecasted. This provides an important tool for informed decision-making and coordinated actions of producers and practitioners to control or mitigate the impact of PEDv outbreaks.
Project #: 19-237 | Principal Investigator: Derald Holtkamp1 | Co-Invesitgators: Clayton Johnson2, Jacek Koziel1, Peiyang Li1, Deb Murray3, Chelsea Ruston1, Aaron Stephan4, Montse Torremorell5, Katie Wedel6
Institutions:1Iowa State University, 2Carthage Veterinary Services, 3New Fashion Pork, 4ONCE, Inc., 5University of Minnesota, 6Iowa Select Farms
UVC germicidal chambers are used in swine settings to reduce the microbial load on surface items. Chambers, which may be commercial or homemade, are usually constructed so items to be disinfected are passed through from the dirty side (entry/hallway) to the clean side (office/break room).
UVC germicidal chambers are mostly used for small to medium items like lunch boxes, cell phones, small tools, and medications. Food and semen bags can also be passed through the chamber without negative effects. Repeat exposure of plastics to UVC light may lead to a change in the color or smell of the object. Paper and cardboard cannot be disinfected in a UVC germicidal chamber. Larger UVC chambers, or UVC rooms, can be built for larger items.
Project #: 19-211 | Principal Investigator: Gustavo Machado1* | Co-Invesitgators: Jason A. Galvis1, Chris Jones2, Cesar A. Corzo3, Joaquin M. Prada4
Institutions: 1 College of Veterinary Medicine, Raleigh, North Carolina, USA.2 North Carolina State University, Raleigh, North Carolina, USA. 3 University of Minnesota, St Paul, MN, USA. 4University of Surrey, Guildford, UK
A limited understanding of transmission dynamics is a major obstacle to prevent and control disease spread among swine populations. Forecasting outbreaks before they occur would allow the swine industry to tailor control strategies and improve pig production. Understanding what combination of strategies may be required to reduce between-farm transmission is key to maintain control of outbreaks while minimizing disruptions to pig production. Our objective is to forecast weekly Porcine Epidemic Diarrhea virus (PEDV) outbreaks by generating high-resolution maps to identify current and future PEDV high risk areas, and simulating the impact of control measures. Three epidemiological transmission models were developed and compared: a novel epidemiological modelling framework was developed specifically to model swine populations, PigSpread and two models built on previously developed ecosystems; SimInf (an stochastic disease spread simulations) and PoPS (Pest or Pathogen Spread). Prediction accuracy across models was compared using receiver operating characteristic (ROC) area under the curve (AUC). The models were calibrated on true weekly PEDV outbreaks from three spatial related swine companies. Model outputs had general agreement with observed outbreaks throughout the study period. PigSpread had an AUC of 0.71, SimInf had an AUC of 0.59 and PoPS had an AUC of 0.80. Our analysis estimates that the combined strategies of herd closure, feedback and reinforcement of on-farm biosecurity reduce 14% the incidence of outbreaks in sow and 20% in Gilt Development Units (GDU) when deployed weekly in sow and GDU farms located in risk areas. Accurate forecasts of PEDV spread are feasible to be generated within a decision making timeframe, but the predictability across all models depends on the stage of the epidemic.
Project #: 19-220 | Principal Investigator: Ganwu Li | Co-Invesitgators: Phillip C. Gauger, Eric Burrough, Jianqiang Zhang, Leyi Wang, Thomas Petznick
Institutions: Iowa State University
A case from the farm experiencing an ongoing problem with piglet diarrhea in the lactation phase for more than 2 years. The pigs exhibited a self-limiting diarrhea starting around 10 days of age, but typically lost 1-2 lbs of expected weaning weight. Histopathological examination of five clinically affected piglets revealed that 5/5 small intestines had moderate villus atrophy, vascular congestion, and lymphocytic infiltration in the small intestine suggestive of an enteric viral infection. Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus, transmissible gastroenteritis virus (TGEV), or rotavirus were not detected from small intestines using real-time PCR assays (rRT-PCR). Additionally, there was no significant bacterial growth from the small intestines. Herein, we document the discovery of porcine sapovirus of genogroup III as the cause of the enteritis and diarrhea use of four independent lines of evidence: metagenomics analysis, real-time RT-PCR, histopathology, and in situ hybridization. To our best knowledge, this is the first evidence that SaV likely serves as the sole etiological agent causing enteritis and diarrhea of piglets in the field in the United States. In addition, a highly sensitive and specific real-time RT-PCR for detecting porcine sapovirus of genogroup III was established. Prevalence survey of more than 500 samples from both pigs with clinical diarrhea and clinical healthy pigs suggests that porcine sapovirus III play an important role in causing swine enteritis and diarrhea and rRT-PCR is a reliable method to evaluate the pathogenicity role of porcine SaV.
Project #: - | Investigators: Orlando Perez1, Mathieu Pinette1, Guang-Zhi Tong2, En-Min Zhou3, Janfa Bai4, Lalitha Peddireddi4, John Schiltz5, Karthik, Shanmuganatham5, Rachel M. Tell5, Aruna Ambagala1 | Institutions: 1National Centre for Foreign Animal Disease, Canadian Food Inspection Agency 2Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 3College of Veterinary Medicine, Northwest A & F University, 4College of Veterinary Medicine, Kansas State University, 5National Veterinary Services Laboratories, VS, APHIS, USDA
Pseudorabies virus (PRV) causes Aujeszky's disease or pseudorabies (PR), fatal encephalitis in newborn piglets, respiratory infection in growing and fattening pigs, and reproductive failures in pregnant sows. It establishes a lifelong latent infection in the peripheral nervous system followed by subsequent intermittent shedding of infectious virus. Since 2011, highly virulent PRV strains that are genetically different from the classic PRV strains surfaced in pig herds in China. Availability of a highly sensitive and specific polymerase chain reaction (PCR)-based diagnostic assay for rapid differential detection of PRV variants is critical to prevent huge economic losses to the U.S. and Canadian pork industries if these strains enter North America and cause an outbreak. Here we describe the development and evaluation of a single-tube triplex real-time-PCR assay for differential detection of variant strains of PRV. The assay targets the intergenic region between the US2 and US6 genes in the PRV genome and is highly sensitive and specific and it did not detect other nontarget viruses including related herpesviruses. The clinical specificity and sensitivity of the assay was evaluated using whole blood, serum, tissue and swab samples collected from known negative and experimentally inoculated pigs with either classical (Bristol) or variant (JS-2012 and HeN1) PRV strains. The targeted genomic region of this assay is also deleted in commonly used PRV gE-deleted marker vaccines, and therefore, the triplex assay did not detect viral DNA extracted from two commercial vaccine strains Bartha K-61 and Bucharest. This single-tube triplex assay can be used for routine diagnostics and epidemiological studies for detection and differentiation of classical strains from variant strains of PRV, and as a differentiation of infected and vaccinated animals (DIVA) assay when PRV gE- deletion mutant marker vaccines are used.
Project #: 19-149 |Principal Investigator: Derald Holtkamp, PhD | Institution: Iowa State University College of Veterinary Medicine
The need to quickly identify, control, and eliminate a pathogen in an endemic, emerging, or transboundary disease outbreak in the United States is crucial to protect the swine industry from suffering huge economic losses. In August of 2016, SHIC funded Iowa State University to develop the Rapid Response to Emerging Disease Program (RRP). A Rapid Response Corp (RRC) was formed for the program. The RRC is a nationwide network of veterinarians, state animal health officials or representatives, epidemiologists and, when appropriate, federal animal health officials who are trained, prepared and committed to moving within 24 hours of contact to conduct epidemiological investigations when a new transboundary or emerging disease threat occurs. The standardized approach, methodology, forms, and reports developed for the Iowa Pork Producers Association (IPPA) funded PRRS outbreak investigation pilot project, were adapted to develop the RRP. Online training modules, checklists, investigation forms, surveys, and report templates to conduct a rapid response investigation are now available for download on the SHIC website. The objective of this project was to facilitate the continuation of the RRP beyond the current five-year funding stream for SHIC by automating and streamlining the rapid response investigation process through a web application. This proposal included the planning phase to automate and streamline the process. The RRC was maintained at 35 members for the duration of this project. To support the continuation of the RRP, and to provide members of the RRC with additional training, a pre-conference seminar was conducted at the American Association of Swine Veterinarian’s Annual Meeting in March of 2020. The seminar was titled “Conducting effective outbreak investigations: Learning from our mistakes, part 2.” The seminar followed the successful pre-conference workshop offered as training for the RRC members at the same meeting in 2019. The possibility of integrating the RRP into the National Pork Board’s swine business continuity system (AgView®) was explored. Delays in the development of AgView led to the exploration of simpler, more executable approaches. A web-based version of the investigation form used by the RRC members to conduct outbreak investigations has been developed using Qualtrix, which is a commercially available online survey platform. The web-based version of the investigation form will be tested in Vietnam, as part of another SHIC funded study to investigate African swine fever (ASF) virus on farms in Vietnam.
Project #: 19-148 |Principal Investigator: Cassandra Jones, PhD | Institution: Kansas State University Department of Animal Sciences & Industry
Environmental monitoring is commonly used in pharmaceutical, human food, and pet food manufacturing facilities manufacturing as an indicator of pathogenic bacteria in the product. A correlation between the presence of Salmonella spp. and Enterobacteriaceae within feed mills has been demonstrated, but little information is available on how the presence of Enterobacteriaceae (EBAC) correlates with viral pathogen presence, especially on farms or in feed mills. The purpose of this study was to identify Enterobacteriaceae presence in the feed manufacturing facilities of a multi-farm system experiencing a viral outbreak as a method of identifying biosecurity gaps.
Project #: 19-219 |Principal Investigators: Derald Holtkamp, DVM, MS | Co-Investigators: Chelsea Ruston, DVM, Daniel Linhares, PhD, MBA, DVM | Institution: Iowa State University College of Veterinary Medicine
The swine industry has focused much of its efforts on improving biosecurity in breeding herds; while little attention had been paid in wean-to-finish growing sites. One risk event that has the potential to introduce virus into grow-finish pigs is load-out during marketing. In order for the remaining pigs in the group to become infected during load-out, viral contamination must be transferred from the contaminated livestock trailer, driver or other carrying agents to the pigs in the barn. Unfortunately, little research has been done to assess how frequently this occurs or to assess alternative biosecurity measures to reduce the frequency.
Project #: 19-236 | Investigators: Xuhua Chen, Nubia Resende-De-Macedo, Panchan Sitthicharoenchai, Orhan Sahin, Eric Burrough, Maria Clavijo,
Rachel Derscheid, Kent Schwartz, Kristina Lantz, Suelee Robbe-Austerman, Rodger Main, Ganwu Li Institution: Iowa State University
High mortality events due to Streptococcus equi subspecies zooepidemicus (Streptococcus zooepidemicus) in swine have not previously been reported in the United States. In September and October 2019, outbreaks with swine mortality up to 50% due to S. zooepidemicus septicaemia were reported in Ohio and Tennessee. Genomic epidemiological analysis revealed that the eight outbreak isolates were clustered together with ATCC 35246, a Chinese strain caused outbreaks with high mortality, also closely related to three isolates from human cases from Virginia, but significantly different from an outbreak-unrelated swine isolate from Arizona and most isolates from other animal species. Comparative genomic analysis on two outbreak isolates and another outbreak-unrelated isolate identified several genomic islands and virulence genes specifically in the outbreak isolates only, which are likely associated with the high mortality observed in the swine population. These findings have implications for understanding, tracking and possibly preventing diseases caused by S. zooepidemicus in swine.PCV3 may cause death in fetuses and myocarditis and systemic vasculitis in pigs.
Project #: 18-201 | Principal Investigator: Albert Rovira | Institution: University of Minnesota
Porcine circovirus type 3 was discovered in 2016 in the US associated with cases of systemic disease and reproductive disorders. Multiple studies performed during the last two years have shown that this virus is widespread and has been around for decades. It can be found in multiple tissues and samples, in pigs with multiple clinical conditions and in healthy pigs. However, we are lacking precise information on the relevance of this virus and its potential to cause disease in pigs. The objective of this proposal was to mine the diagnostic data obtained by the MN VDL during the last 2 years to identify associations between the presence of PCV3 and its viral load and specific lesions and clinical conditions. PCV3 results, clinical signs and information on the lesions for each pig were retrieved from the MN VDL laboratory information management system, submission forms and diagnostic reports. PCV3 frequency in pigs with different clinical signs ranged from 12% to 27%. No significant associations were observed between clinical signs and the presence of PCV3. In PCV3-positive pigs, no clinical signs were significantly associated with having a higher load of PCV3. PCV3 frequency in pigs with different lesions ranged from 0% to 62%. The only lesion that had a significant association between its presence and PCV3 infection was heart vasculitis/perivascultis. In PCV3-positive pigs, higher viral loads were significantly associated with pigs with myocarditis, heart vasculitis/ perivasculitis, kidney vasculitis/perivasculitis and dermatitis. This study did not identify any significant associations with clinical signs. However, the presence of PCV3 in 20% of fetuses is remarkable. In addition, the samples with the highest PCV3 concentration in this study were from fetal tissues. Lesions of myocarditis and systemic vasculitis were associated with the presence or the amount of PCV3 in tissues. The lack of significant results for other lesions does not exclude the possibility of a real association and may be due to confounding factors or limited data. In summary, this study provides an objective view of the relationship between PCV3 and disease, based on a large dataset of diagnostic cases. PCV3 is a very common virus that circulates in healthy populations and can be detected in around 20% of the pigs submitted to the diagnostic laboratory. Therefore, it is important to differentiate when PCV3 plays a significant role and when it does not. The results from this study support previous studies that suggested that PCV3 may cause death in fetuses and myocarditis and systemic vasculitis in pigs.
Project #: 20-072 | Principal Investigator: Scott Dee | Institution: Pipestone Applied Research
In 2014, contaminated feed was identified as a vehicle for the transport and transmission of PEDV. Over time, similar conclusions were drawn regarding the role of feed and the risk of African swine fever virus, Senecavirus A (SVA), Pseudorabies virus and Classical swine fever virus. However, as these results were based on laboratory studies, our goal was to validate these observations using a real-world “demonstration project” approach, simulating the actual transport of feed contaminated with SVA, PEDV and PRRSV across the United States in a commercial transport vehicle. Samples of conventional soybean meal, organic soybean meal, lysine, choline and vitamin A were spiked with all 3 viruses and placed in a trailer of a commercial transport vehicle. The samples were stored in vented containers to allow exposure to environmental factors including temperature and relative humidity during the trip. Overall, the journey involved 21 days, 107 hours of transport, crossed 14 states and covered approximately 6000 miles. At the end of the study period, samples were tested for the presence of viral genome by PCR and viable virus by swine bioassay. Results indicated the presence of viable PRRSV, SVA and PEDV in both soy products, while viable SVA was recovered from all 5 ingredients. In contrast, survival was limited in the vitamins and amino acid ingredients. These results validate published laboratory data indicating the protective nature of soy-based feed ingredients and demonstrate that certain viruses, such as SVA are very stable in feed. In closing, this novel approach did demonstrate that three significant viral pathogens of pigs could survive in select feed ingredients during an actual shipping event, involving diverse environmental conditions and realistic transit periods. It is hoped that the information derived from this study will help to unify opinions across the swine industry, the veterinary profession, and governmental agencies regarding the risk of feed.
Project #: 16-262 | Principal Investigator: Rodger Main | Co Investigators: Mike Martin, Gary Anderson, Jane Hennings, Jerry Torrison, Stephanie Rossow, Christina Loiacono, Sarah Tomlinson | Institutions: Iowa State University Veterinary Diagnostic Laboratory, Clemson University, Kansas State University Veterinary Diagnostic Laboratory, South Dakota State Animal Disease Research and Diagnostic Laboratory, University of Minnesota Veterinary Diagnostic Laboratory, USDA NAHLN
Seamless integration of diagnostic data from any number of veterinary diagnostic laboratories (VDLs) into third-party database applications for further analytical and reporting purposes has long been recognized as a critical element necessary for proficient detection, monitoring, response, and/or management of significant diseases across a region, state, or nation. Establishing and adopting the use of universally recognized diagnostic data standards and a common electronic messaging schema are foundational elements necessary for the development of sustainable and scalable systems of connectivity and web-based analytical tools necessary to support the current needs and future demands of the US Pork Industry.
This highly collaborative initiative served to further develop the core infrastructure and diagnostic data standards necessary for US Pork Industry stakeholders to effectively harness the capabilities that any number of existing or yet to be developed (web-based) animal health information management technologies aim to provide. Primary deliverables included the development of a more comprehensive electronic message schema; a greatly expanded formulary of now more than 700 standardized test result codes covering the full-spectrum of diagnostic tests commonly conducted on swine case submissions to veterinary diagnostic labs (VDLs); an intuitive, web-based search engine that enables users (e.g., VDL information technology personnel) to readily identify the appropriate diagnostic test result code(s) (i.e., Logical Observation Identifier Names and Codes, or LOINC®) to use for each of the specific assays conducted and reported from their laboratory; and a web-based electronic message validator that allows VDLs to test, trouble-shoot, and validate their electronic messaging capabilities.
Each of the VDLs collaborating on this infrastructure development project demonstrated their ability to electronically synthesize and successfully deliver results from diagnostic case submissions utilizing the updated electronic messaging schema and expanded formulary of diagnostic test result codes. To ensure national level scalability, the updated electronic messaging schema derived from this project is consistent and fits within the overarching architecture of the Health Level Seven® (HL7) messaging schema adopted by the United States Department of Agriculture National Animal Health Laboratory Network (USDA NAHLN). The expanded formulary of standardized codes for diagnostic test results (LOINCs); the updated and more comprehensive HL7 electronic messaging schema; the standardized diagnostic test result code (LOINC®) search engine; and the HL7 electronic message validator application developed as a result of this project will be made available for use by VDLs across the USDA NAHLN.
The infrastructure developed in accordance with the primary deliverables of this initiative will provide a lasting foundation for enhancing the adoption and use of universally recognized diagnostic data standards and HL7 electronic messaging capabilities in swine interest VDLs in the USDA NAHLN. Furthering the establishment and use of such standards are critical building blocks for enhancing the connectivity and interoperability of diagnostic information across VDLs and for broadly transcending swine diagnostic information management into the digital era. Albeit these developments centered at the four VDLs collaborating on this project and were specifically focused on diagnostic tests conducted on US swine, this precedent-setting work could readily be expanded and emulated for use across any number of animal species and VDLs.
Project #: 18-137 | Principal Investigator: Diego G. Diel | Institution: South Dakota State University
Feed biosecurity became a topic of much interest to the swine industry, given recent results suggesting that feed can harbor viable viral pathogens and potentially serve as source of infection to susceptible pigs. The goal of this study was to evaluate the mitigation potential of chemical feed additives following natural consumption of contaminated and mitigated feed. To determine whether chemical mitigation of feed could reduce or prevent pathogen transmission through feed, we performed a feed trial experiment in which animals were allowed to ingest contaminated or contaminated and mitigated feed for three consecutive days. After feeding, each animal was sampled individually and levels of viremia, virus shedding and viral load in tissues were determined by RT-qPCR. For this in vivo trial we selected three candidate mitigants, A, B, and C, from our previous in vitro mitigation project. Results of our trial show that only mitigant A reduced Senecavirus A transmission through feed. Whereas no significant differences between control non-mitigated and mitigated feed were observed for porcine epidemic diarrhea virus. Results here, under conditions in which each animal ingested contaminated and mitigated feed, show that that chemical mitigation alone (with mitigants A, B, and C) may not be able to prevent transmission of pathogens through feed. These findings can be likely attributed to many factors, including: 1) the dose of virus used in our trial; 2) the fact that the chemical mitigants don’t reduce viral load in feed to levels that are non-infectious to susceptible pigs; and 3) poor contact time of the mitigant with the virus. Therefore, alternative strategies such as storage time and importation of feed ingredients from known and trusted sources should also be carefully considered to safeguard the US swine industry from unwanted viral pathogens that are endemic in other regions of the world. Additionally, studies on the mechanism of action of potential mitigants may also allow selection of those compounds that present the greatest chance of virus inactivation in the feed matrix.
Project #: 18-215 | Principal Investigator: Luis Giménez-Lirola | Institution: Iowa State University
This research represents a collaborative effort between researchers at the Canadian Food Inspection Agency (CFIA), USDA Agricultural Research Service (ARS), and Iowa State University.
Background Beginning in the 1960s, clinical PRV became increasingly problematic in commercial swine herds in Europe, the Americas, and Southeast Asia, with annual losses estimated at $21 to $25 million (USD) to U.S. producers (Miller et al., 1996; Neumann et al., 2005). The U.S. officially eliminated PRV from domestic swine in 2004, but PRV is occasionally introduced into "transitional" herds via contact with feral swine, e.g., Minnesota (2002) and Wisconsin (2007).
Ultimately, PRV control and elimination was achieved by exploiting the molecular biology of the virus in a PRV DIVA (differentiation of infected from vaccinated animals) strategy (Quint et al., 1987; van Oirschot et al., 1986). In brief, the PRV viral envelope includes up to 11 glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, gL, gM, gN) (Mettenleiter, 2000). Relevant to PRV control, glycoprotein gB is highly conserved and required for both viral replication and cell-to-cell spread of the virus. In contrast, gE is not required for PRV replication and is associated with virulence. Wild-type viruses contain both gB and gE, while PRV MLV vaccines contain gB, but not gE. Thus, the genetic profile of vaccine virus is gB+/gE- while wild-type virus is positive for both genes (gB+/gE+). This clear distinction between wild-type and MLV viruses has allowed for the development of a DIVA approach to PRV control and eradication. That is, gene-deleted vaccines are used to protect animals against clinical PRV, with differential antibody ELISAs or DNA PCRs used to detect infected animals.
In 2018, PRV was listed as #4 in the SHIC swine disease matrix because of the potential for introducing highly pathogenic PRV into the US from Asia (https://www.swinehealth.org/swine-disease-matrix/) and its potential negative impact on exports. For this reason, improvements in PRV diagnostics, surveillance, control, and elimination remain relevant.
Project objectives The PRV PCRs have been developed for individual animal nasal swabs, but not for swine oral fluid. Therefore, the objective of this project was to evaluate the detection of PRV in swine oral fluid collected from vaccinated and/or inoculated pigs using two contemporary real-time PCR assays targeting PRV gB and gE genes.
Detection of PRV in oral fluids and nasal swabs Diagnostic samples of precisely known PRV infection status were used to evaluate assays and establish shedding dynamics. In this project, nasal swabs and oral fluid samples were collected from forty 12- to 16-week-old pigs in 4 treatment groups (10 pigs per group): PRV vaccinated with MLV and challenged with classical wild-type virus at 20 days post vaccination (DPV) (Group 1), PRV vaccinated (Group 2), wild-type PRV inoculated (Group 3), and negative control (Group 4). To detect the presence of PRV DNA, samples were tested using 1) gB PCR for screening PRV-positive animals and 2) gE triplex PCR for differentiation between PRV classic and PRV high-pathogenic (China) strains.
Results PRV DNA was detected by 0 DPV and 1 DPI in oral fluid and nasal swab samples from vaccinated and/or wild-type virus inoculated animals using the gB screening PCR. The detection of vaccine and wild-type viruses were up to 2 and 30 days, respectively. The post-inoculation detection rate was lower with shorter shedding period in vaccinated pigs (Group 1). The shedding patterns in oral fluid were comparable to which in nasal swabs. Four false positive results were observed in nasal swabs from vaccinated pigs (Group 1 and 2). The gE triplex PCR likewise detected classical strain PRV in both oral fluid and nasal swab samples from inoculated pigs (Group 3), but at a lower frequency than the gB PCR.
Conclusions This study showed that PRV DNA could be detected in swine oral fluid specimens using PRV gB and gE real-time PCRs. Furthermore, comparisons of detection rates in nasal swab vs oral fluid samples suggested that oral fluid could be used as an alternative to individual pig (nasal swab) sampling for PRV surveillance. However, further improvements in the performance of both the gB and gE PCRs would be recommended.
Contact info: Dr. Luis Gimenez-Lirola, Iowa State University, College of Veterinary Medicine, 1800 Christensen Drive, Ames, IA. Email: email@example.com. Phone: 1-515-294-7025
Project #: 18-146 | Principal Investigator: Diego G. Diel | Institution: Cornell University
The swine acute diarrhea syndrome coronavirus (SADS-CoV) RT-PCR assay was incorporated into the EZ-PED/TGE/PDCoV MPX 1.1 RT-PCR assay. The SADS RT-PCR assay replaced the TGE RT-PCR assay in the multiplex. A comparison was completed between EZ-PED/TGE/PDCoV MPX 1.1 vs EZ-PED/SADS/PDCoV MPZ. Swine fecal and oral fluid samples were randomly selected and extracted for testing with both assays side-by-side. As shown in the report, results between the two assays was very similar, indicating that the newly developed SADS-CoV is compatible and does not interfere with the PED and PDCoV assays.
The new multiplex real-time PCR (EZ-PED/SADS/PDCoV) was evaluated in China using samples with known enteric and SADS coronavirus status. This portion of the project was carried out in the laboratory of Dr. Shao Lun Zhai at the Guangdong Academy of Agricultural Sciences. All the samples tested with the new multiplex PCR had been previously tested with an in-house PCR developed at Dr. Zhai's laboratory. As shown in the report, overall results from Dr Zhai's PCR and the newly developed assay here are consistent, with the newly developed assay showing increased capability of detecting a few additional positive samples for all three pathogens (PEDV, PoDV and SADS-CoV) when compared to the in-house PCR. An interesting observation of these data is the fact that most SADS-CoV positive samples were also positive for PEDV or PDCoV. Some samples were also positive for all three pathogens.
Project #: 18-213 | Principal Investigator: Cesar A Corzo | Institution: University of Minnesota
Objective 1: Monitor trends in pathogens incidence and prevalence – PRRSv, PEDv, PDCoV, Senecavirus and central nervous system associated viruses continue to be monitored in time and space. The 2018-2019 season fortunately ended with the second lowest PRRSv breeding herd cumulative incidence (21.3%) during the last 10 years of monitoring. Still, herds that break remain in category 1 for longer periods. PEDv also followed the same trend and maintained a low level of cumulative incidence with the few cases being reported throughout the country. PDCoV continues to be present in our industry clustered in specific regions; however, incidence is low as cases of this disease are not reported frequently.
Objective 2: To conduct prospective monitoring of PRRSv sequence evolution and impact – PRRSv sequence capturing process and analysis continues and our database currently has 30,603 sequences from 31 systems. Preliminary analysis showed that PRRSv sequences cluster in time and space as expected. Similarity analysis was conducted to understand how viruses have changed when compared to the reference strain VR2332. Majority of viruses are 10-15% different while there is a subset of viruses that lie between 2-5%. A similar analysis was conducted for viruses that have been classified as 1-7-4 as these viruses by comparing them to the first reported virus of this RFLP group. Diversity among newly reported 174 viruses appear to be decreasing through time together with the occurrence of this virus.
Objective 3: Develop capacity to capture and analyze movement data – The new implemented technology has been generating truck movement data consistently. A total of 12 vehicles (8 trucks and 4 trailers) are being monitored in one of the SHMP participating systems. Data structure and processes for downloading data are still being understood and procedures developed to facilitate the data analysis. A first attempt was made and transport networks were being built to understand truck movement and frequency. As expected, the data shows that movements between sow farms-growing pig sites-trucks washes are the most frequent.
Objective 4: To expand participation of producers to allow for all to be involved – Expansion continues as 2 new production companies joined the project for a total of 38. The search for new potential participants will continue. As the SQL database has enable the capability to expand in the number of farms to be added to our database, the addition of growing pig sites from specific participants is in process as we have received authorization to move forward.
Project #: 18-198 | Principal Investigator: J Zimmerman, C Wang, R Main | Institution: Iowa State University
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.
Project #: 16-273 and 19-154 | Principal Investigator: Gustavo S. Silva | Institution: Iowa State University
Investments in biosecurity practices are made by producers to reduce the likelihood of introducing pathogens such as porcine reproductive and respiratory syndrome virus (PRRSv). The assessment of biosecurity practices in breeding herds is usually done through surveys. The objective of this study was to evaluate the use of machinelearning (ML) algorithms to identify key biosecurity practices and factors associated with breeding herds selfreporting (yes or no) a PRRS outbreak in the past 5 years. In addition, we explored the use of the positive predictive value (PPV) of these models as an indicator of risk for PRRSv introduction by comparing PPV and the frequency of PRRS outbreaks reported by the herds in the last 5 years. Data from a case control study that assessed biosecurity practices and factors using a survey in 84 breeding herds in U.S. from 14 production systems were used. Two methods were developed, method A identified 20 variables and accurately classified farms that had reported a PRRS outbreak in the previous 5 years 76% of the time. Method B identified six variables which 5 of these had already been selected by model A, although model B outperformed the former model with an accuracy of 80%. Selected variables were related to the frequency of risk events in the farm, swine density around the farm, farm characteristics, and operational connections to other farms. The PPVs for methods A and B were highly correlated to the frequency of PRRSv outbreaks reported by the farms in the last 5 years (Pearson r=0.71 and 0.77, respectively). Our proposed methodology has the potential to facilitate producer’s and veterinarian’s decisions while enhancing biosecurity, benchmarking key biosecurity practices and factors, identifying sites at relatively higher risk of PRRSv introduction to better manage the risk of pathogen introduction.
Project #: 18-24 | Principal Investigators: Dr. Cassandra Jones, et. al. | Institution: Kansas State University
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.
Project #: 17-189 | Principal Investigator: Megan Niederwerder, DVM, PhD | Institution: Kansas State University
African swine fever (ASF) is currently considered the most significant global threat to pork production worldwide. Disease caused by the ASF virus (ASFV) results in high case fatality of pigs. Importantly, ASF is a trade-limiting disease with substantial implications on both global pork and agricultural feed commodities. ASFV is transmissible through natural consumption of contaminated swine feed and is broadly stable across a wide range of commonly imported feed ingredients and conditions. The objective of the current study was to investigate the efficacy of medium-chain fatty acid and formaldehyde-based feed additives in inactivating ASFV. Feed additives were tested in cell culture and in feed ingredients under a transoceanic shipment model. Both chemical additives reduced ASFV infectivity in a dose-dependent manner. This study provides evidence that chemical feed additives may potentially serve as mitigants for reducing the risk of ASFV introduction and transmission through
Project #: 18-197 | Principal 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.
Project #: 18-189 | Principal 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.
Project #: 18-136 | Principal Investigator: Bailey Arruda | Institution: Iowa State University
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.
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
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.
Project #: 18-193 | Investigator: Cesar A Corzo | Institution: University of Minnesota
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.
Project #: 17-199 | Investigator: Douglas Marthaler | Institution: Kansas State University
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.
Project #: 17-141 | Investigator: Jeff Zimmerman, DVM PhD | Institution: Iowa State University
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.
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.
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.
Project #: 18-211 | Investigator: Diego G. Diel | Institution: South Dakota State University
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.
Project #: 17-187 | Investigators: Diego G. Diel and Scott Dee | Institution: South Dakota State University
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.
Project #: - | Investigators: Gustavo S. Silva, Luis G. Corbellini, Daniel L.C. Linharea, Kimberlee L. Baker, Derald J. Holtkamp | Institutions: Iowa State University and Universidade Federal do Rio Grande do Sul
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.
Project #: 17-188 | Investigator: Cassandra Jones | Institution: Kansas State University
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.
Project #: 17-191 | Investigators: J. Zimmerman, DVM PhD, L. Giménez-Lirola, PhD, K. Poonsuk, DVM PhD. | 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.
Project #: 16-250 | Principal Investigator: Aruna Ambagala | Collaborators: Guang-Zhi Tong, En-Min Zhou, Jianfa Bai, Lalitha Peddireddi, John Schiltz, Sabrina Swenson, Karthik K Shanmuganatham | Institution: National Centre for Foreign Animal Disease- Winnipeg, MB
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.
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
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.
Project #: 17-144 | Principal Investigators: Dr. Phil Gauger DVM, PhD, Associate Professor; Dr. Karen Harmon, PhD, Clinical Associate Professor | Institution: Iowa State University
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; firstname.lastname@example.org
Project #: 16-273 | Principal Investigator: Daniel Linhares | Co Investigators: Gustavo Silva, Kimberlee Baker, Derald Holtkamp, Bob Morrison | Institutions: Iowa State University, University of Minnesota
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.
Project #: 16-256 | Principal Investigator: Lalitha Peddireddi | Institutions: Kansas State Veterinary Diagnostic Laboratory, Kansas State University
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; email@example.com.
Project #: 16-175 | Principal Investigator: Daniel Linhares | Co-Investigators: Jeff Zimmerman & Marcelo Almeida | Institution: Iowa State University
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.
Project #: 16-257 | Principal Investigator: Jianfa Bai | Institution: Kansas State University
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; firstname.lastname@example.org.
Project #: 16-275 | Principal Investigators: Benjamin Blair and James Lowe | Institutions: Integrated Food Animal Medicine Systems, Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign
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.
Project #: 15-195 | Principal Investigators: Pablo Pineyro, DVM PhD and Luis Gimenez-Lirola, PhD | Institution: Iowa State University
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.
Project #: 15-206 | Principal Investigator: Dr. Chris Rademacher | Institution: Iowa State University
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.
Project #: 15-192 | Principal Investigators: Diego G. Diel | Co-Investigators: Travis Clement, Eric Nelson, Jane Hennings, Steven Lawson, Luizinho Caron, Rejane Schaefer | Institutions: South Dakota State University, EMBRAPA Swine and Poultry
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.
Project #: 15-188 | Principal Investigators: Steven Lawson | Co-Investigators: E. Nelson, D. Diel, A. Singrey, T. Clement, J. Christopher-Hennings | Institution: South Dakota State University
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:
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.
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.
Project #: 15-195 | Principal Investigators: Pablo Pineyro, DVM PhD and Luis Gimenez-Lirola, PhD | Institution: Iowa State University
The specific aims of this proposal are to develop a set of direct diagnostic tools that allows direct detection of SV-A in situ.
Project #: 15-195 | Principal Investigators: Pablo Pineyro, DVM PhD and Luis Gimenez-Lirola, PhD | Institution: Iowa State University
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.
Project #: 15-181 | Principal Investigator: James Roth
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.
Project #: 15-185 | Principal Investigators: Main R, Rossow S, Gauger P, Harmon K, Marthaler D, Vannucci F, Zhang J.
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.
Project #: 15-187 | Principal Investigator: Goyal, Sagar M.
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).
Project #: - | Principal Investigator: Holtkamp, Derald, et al.
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).
Project #: 15-193 | Principal Investigator: Diel, Diego G.
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.
Project #: 15-199 | Principal Investigator: Tousignant, Steve, et al.
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.
Project #: 15-199 | Principal Investigator: Tousignant, Steve, et al.
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.