An Evaluation of Porcine Epidemic Diarrhea Virus Survival in Individual Feed Ingredients in the Presence or Absence of a Liquid Antimicrobial

Background: Contaminated complete feed and porcine plasma are risk factors for PEDV introduction to farms and a liquid antimicrobial has been proven useful for reducing risk. This study provides information on the survivability of PEDV across common swine feed ingredients in the presence or absence of the liquid antimicrobial.Results: Eighteen ingredients commonly included in commercial swine diets were selected, including 3 grain sources (corn, soybean meal (SBM), dried distillers grains with solubles (DDGS)), 5 porcine by-products (spray-dried plasma, purified plasma, intestinal mucosa, meat and bone meal and red blood cells (RBCs)), 3 vitamin/trace mineral (VTM) mixes (sow, nursery, finishing), 2 fat sources (choice white grease and soy oil), 3 synthetic amino acids (lysine HCL, D/L methionine, threonine), as well as limestone and dry choline chloride. Complete feed and stock PEDV served as controls. Thirty grams of each ingredient were inoculated with 2 mL PEDV. A matched set of samples were treated with the formaldehyde-based liquid antimicrobial SalCURB® (LA). All samples (n = 320) were stored outdoors under winter time ambient conditions for 30 days. Samples were submitted on 1, 7, 14 and 30 days post-inoculation (DPI) and tested by PCR and virus isolation (VI). All VI-negative samples were tested by swine bioassay. Viable PEDV was detected by VI or swine bioassay at 1, 7, 14 and 30 DPI from SBM, DDGS, meat & bone meal, RBCs, lysine HCL, D/L methionine, choice white grease, choline chloride, complete feed and stock virus control and at 7 DPI in limestone and at 14 DPI in threonine. Supplementary testing of complete feed and SBM indicated viable virus out to 45 and 180 DPI, respectively. All other samples were negative by VI and bioassay. In contrast, treatment with LA inactivated PEDV across all ingredients on 1 DPI and induced RNA reduction over time.Conclusions: Under the conditions of this study, PEDV viability in feed was influenced by ingredient with extended survival in SBM. Furthermore, LA treatment rendered virus inactive, independent of ingredient type

Development of an Indirect ELISA, Blocking ELISA, Fluorescent Microsphere Immunoassay and Fluorescent Focus Neutralization Assay for Serologic Evaluation of Exposure to North American Strains of Porcine Epidemic Diarrhea Virus

Recent, severe outbreaks of porcine epidemic diarrhea virus (PEDV) in Asia and North America highlight the need for well-validated diagnostic tests for the identification of PEDV infected animals and evaluation of their immune status to this virus. PEDV was first detected in the U.S. in May 2013 and spread rapidly across the country. Some serological assays for PEDV have been previously described, but few were readily available in the U.S. Several U.S. laboratories quickly developed indirect fluorescent antibody (IFA) assays for the detection of antibodies to PEDV in swine serum, indicating prior exposure. However, the IFA has several disadvantages, including low throughput and relatively subjective interpretation. Different serologic test formats have advantages and disadvantages, depending on the questions being asked, so a full repertoire of tests is useful. Therefore, the objective of this study was to develop and validate multiple improved serological assays for PEDV, including an indirect ELISA (iELISA); a highly specific monoclonal antibody-based blocking ELISA (bELISA); fluorescent microsphere immunoassays (FMIA) that can be multiplexed to monitor exposure to multiple antigens and pathogens simultaneously; and a fluorescent focus neutralization assay (FFN) to measure functional virus neutralizing antibodies

Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed

Research has demonstrated that swine feed can be a fomite for viral transmission and feed additives can reduce viral contamination. Therefore, the objective of this study was to evaluate two feed additives in feed contaminated with PEDV or PRRSV. Feed additives included: no treatment, 0.33% commercial formaldehyde-based product, and 0.50% medium chain fatty acids (MCFA) blend. Feed samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106 TCID50/g for each virus. Once inoculated, feed was stored at room temperature for 24 h before analyzing via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a quantitative real time reverse transcription PCR (qRT-PCR) assay was used, which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to detect PEDV and PRRSV within a single assay. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P = 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P \u3c 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P \u3c 0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both feed additives increased Ct in feed when co-inoculated with PRRSV and PEDV. This study also provided evidence that the co-inoculation model can effectively evaluate mitigants

Feed Mitigant Efficacy for Control of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus when Inoculated Alone or Together in Feed

Research has demonstrated that swine feed can be a fomite for viral transmission and feed additives can reduce viral contamination. Therefore, the objective of this study was to evaluate two feed additives in feed contaminated with PEDV or PRRSV. Feed additives included: no treatment, 0.33% commercial formaldehyde-based product, and 0.50% medium chain fatty acids (MCFA) blend. Feed samples were inoculated with PEDV and PRRSV alone or together at an inoculation concentration of 106 TCID50/g for each virus. Once inoculated, feed was stored at room temperature for 24 h before analyzing via qRT-PCR. For samples inoculated with PEDV or PRRSV alone, a quantitative real time reverse transcription PCR (qRT-PCR) assay was used, which was designed to detect PEDV or PRRSV nucleic acid. For co-inoculated samples, an assay was designed to detect PEDV and PRRSV within a single assay. For PEDV alone, there was marginally significant evidence that feed additives resulted in differences in cycle threshold (Ct) value (P = 0.052), but no evidence was observed for pairwise differences. For PRRSV alone, formaldehyde increased Ct compared to the untreated control and MCFA treatment (P \u3c 0.05). For co-infection of PRRSV and PEDV, MCFA and formaldehyde increased Ct (P \u3c 0.05) in comparison to non-treated feed. In summary, formaldehyde increased Ct values in feed when contaminated with PRRSV while both feed additives increased Ct in feed when co-inoculated with PRRSV and PEDV. This study also provided evidence that the co-inoculation model can effectively evaluate mitigants

Understanding the Reduction of Porcine Epidemic Diarrhea Virus, Porcine Reproductive and Respiratory Syndrome Virus, and Seneca Valley Virus 1 RNA in Inoculated Feed and the Environment Following Thermal Processing

Pelleting of feed has been demonstrated to be an effective mitigation strategy for porcine epidemic diarrhea virus (PEDV) contaminated feed but has not been evaluated for other endemic swine viruses like porcine reproductive and respiratory syndrome virus (PRRSV) or Seneca Valley virus 1 (SVV1). Therefore, the objective of this experiment was to evaluate the efficacy of pelleting to inactivate PEDV, PRRSV, and SVV1 inoculated feed. Ten replicates were conducted in the Cargill Feed Safety Research Center at Kansas State University (K-State) using a pilot scale mixer, bucket elevator, pellet mill (including conditioner and die), and cooler. First, a virus negative batch of gestation feed was run through all equipment to simulate a commercial feed mill, then a positive batch of feed inoculated with all three viruses was run through all feed manufacturing equipment. Feed was conditioned to a minimum of 180°F with a 30 sec retention time; all feed was cooled for 10 min. Feed and environmental samples were taken from each piece of equipment following both the negative and positive batch. Samples were analyzed via PCR at the K-State Veterinary Diagnostic Laboratory. A four-room bioassay was conducted to evaluate the infectivity of the feed samples. Feed from the mixer and bucket elevator had greater quantities of SVV1, PEDV, and PRRSV RNA (P \u3c 0.05) than the other sampling locations. Similarly, environmental samples from the mixer and bucket elevator had greater SVV1 detection (P \u3c 0.05) than those collected from the conditioner, pellet die, and cooler. Pelleting reduced viral RNA (P \u3c 0.05) for all viruses in both feed and environmental samples. Although SVV1 and PEDV RNA were still detectable following pelleting, no pigs inoculated with the pelleted feed showed signs of SVV1 or PEDV clinical infection. Interestingly, PRRSV RNA was not detectable in pelleted feed samples. However, one pig showed signs of replicating PRRSV virus on d 7 of the bioassay which suggests a greater sensitivity when utilizing a bioassay compared to PCR alone. Overall, pelleting reduced the quantity of detectable viral RNA and reduced the risk of infectivity; yet small quantities of viral RNA remaining in the feed and environment following pelleting may increase the risk of re-contamination

Evaluating the Distribution of Porcine Epidemic Diarrhea Virus, Porcine Reproductive and Respiratory Syndrome Virus, and Seneca Valley Virus 1 Inoculated Feed After the Use of Physical or Chemical Mitigants to Flush a Feed Manufacturing Facility

Contaminated feed is a route of virus transmission between feed mills and swine farms. To reduce the risk of transmission, an understanding of the virus distribution and mitigation strategies are needed. The objective of this study was to evaluate the distribution of porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), and Seneca Valley virus 1 (SVV1) inoculated feed in the environment and feed of a feed mill before and after the use of chemical mitigants. A 50-lb batch of feed was run through a mixer and bucket elevator followed by a batch inoculated with PEDV, PRRSV, and SVV1. Following the virus-inoculated batch, a flush treatment of either 1) ground corn (GC); 2) GC + 1.5% liquid formaldehyde (LF; SalCURB LF Liquid, Kemin, Des Moines, IA); 3) GC + 1.5% LF + 25% abrasive material (SalCURB; Shell & Bone Builder, Iowa Limestone Company, Urbandale, IA); 4) double flush – GC + 25% abrasive material followed by GC +1.5% LF (Shell & Bone Builder; SalCURB); or 5) dry formaldehyde (SalCURB F2 Dry, Kemin, Des Moines, IA) was utilized, followed by 3 virus-free batches of complete feed. Feed and environmental samples were collected from each piece of equipment following every batch. Dust samples were collected after manufacturing from the inoculated, flush, and final batches from non-feed contact surfaces. Non-feed contact surfaces were considered those where dust would accumulate during manufacturing but would not be included in the final diet. The surfaces included the grates of the mixer, the top of the discharge bin following the bucket elevator, and the floor surrounding the same discharge bin. Samples were analyzed via a triplex PCR at the Kansas State University Veterinary Diagnostic Laboratory. A treatment × batch × location interaction was not observed (P \u3e 0.05) in feed or the environment for any of the viruses. A flush treatment × batch interaction was observed for SVV1 where greater quantities of viral RNA (P \u3c 0.05) were present in the positive batches and the ground corn flush than in those batches which used chemical mitigants or the post-flush batches. A lower quantity of viral RNA (P\u3c 0.05) in dust was observed in the last batch of feed compared to the inoculated batch for all viruses; however, SVV1 RNA was still detectable in the dust following the last batch in all treatments. A batch effect (P \u3c 0.05) was observed in all sample matrices for PEDV and PRRSV as viral RNA decreased after the implementation of the flush regardless of treatment. The use of chemical mitigants and the implementation of a flush batch reduced the quantity of viral RNA for PEDV, PRRSV, and SVV1. However, viral presence was still observed in feed and the dust on non-feed contact surfaces which could be a source of contamination if re-introduced into finished feed

Serological Evidence for the Presence of Influenza D Virus in Small Ruminants

Influenza D virus (FLUDV) was isolated from diseased pigs with respiratory disease symptoms in 2011, and since then the new virus has also been spread to cattle. Little is known about the susceptibility of other agricultural animals and poultry to FLUDV. This study was designed to determine if other farm animals such as goats, sheep, chickens, and turkey are possible hosts to this newly emerging influenza virus. 648 goat and sheep serum samples and 250 chicken and turkey serum samples were collected from 141 small ruminant and 25 poultry farms from different geographical locations in the United States and Canada. Serum samples were examined using the hemagglutination inhibition (HI) assay and the sheep and goat samples were further analyzed using the serum neutralization assay. Results of this study showed FLUDV antibodies were detected in 13.5% (17/126) of the sampled sheep farms, and 5.2% (29/557) of tested sheep serum samples were positive for FLUDV antibodies. For the goat results, the FLUDV antibodies were detected in 13.3% (2/15) of the sampled farms, and 8.8% (8/91) of the tested goat serum samples were positive for FLUDV antibodies. Furthermore, all tested poultry serum samples were negative for FLUDV antibodies. Our data demonstrated that sheep and goat are susceptible to FLUDV virus and multiple states in U.S. have this virus infection already in these two species. This new finding highlights a need for future surveillance of FLUDV virus in small ruminants toward better understanding both the origin and natural reservoir of this new virus