Characterization of porcine circovirus type 2a and 2b infection and lesions in gnotobiotic pigs

Porcine circovirus (PCV) is a member of the genus Circovirus of the Circoviridae family. This family consists of a diverse group of animal viruses that possess a small, closed circular, single-stranded DNA genome that replicates through double-stranded intermediates. Two genotypes of PCV have been identified. PCV type 1 (PCV1) is non-pathogenic, while PCV type 2 (PCV2) has been implicated as the etiological agent of a collection of disease syndromes labeled porcine circovirus associated disease (PCVAD) which include postweaning multisystemic wasting syndrome (PMWS) in swine.;In late 2005, a postweaning, high mortality syndrome spread rapidly through fattening barns in swine dense areas of the United States. Diagnostic investigations consistently isolated porcine circovirus type 2 (PCV2) from diseased tissues. Subsequent genetic analysis revealed the infectious agent was PCV2b. Prior to late 2004, only PCV2a, but not PCV2b, had been reported in North America. In the first manuscript in this thesis, we demonstrate an acute high mortality disease in germ-free pigs using infectious viruses generated from DNA clones constructed from PCV2a and PCV2b isolates identified in the 2005 outbreak. Clinical signs exhibited by diseased pigs included anorexia, dyspnea and listlessness. Mortality was typically observed within 12 hours of onset of dyspnea. The most striking microscopic lesions in affected animals were severe hepatic necrosis and depletion of germinal centers in lymph nodes with associated abundant PCV2 viral antigen. Clinical signs and lesions observed in this study were comparable to those reported in experiments with germ-free pigs inoculated with a PCV2a isolate while concurrently receiving immune-stimulation or co-infection with porcine parvovirus. The animals in this study were confirmed to be free of detectable porcine parvovirus, bovine viral diarrhea virus, porcine reproductive and respiratory syndrome virus, porcine hepatitis E virus, and aerobic and anaerobic bacteria. Thus, in these studies, both PCV2a and PCV2b isolates were singularly capable of inducing high mortality in the absence of any recognized infectious co-factor in the gnotobiotic model.;Porcine circovirus associated disease is a disease complex affecting swine around the world. Although PCV2 is believed to negatively affect the host\u27s immune system, the mechanism by which PCV2 induces disease is not completely understood. The second manuscript in this thesis describes a series of PCV2 experiments using the gnotobiotic pig model in which a correlation was noted between abnormal leukograms and clinical disease in PCV2-infected pigs. The leukogram was characterized by a lymphopenia beginning within 14 days post inoculation (dpi) followed by an absolute neutrophilia approximately one week later. No significant changes in the circulating monocyte, basophil, and eosinophil cell populations were detected. The combination of an absolute neutrophilia and lymphopenia produced a neutrophil/lymphocyte ratio that was predictive of clinical disease and was inversely correlated with the presence of neutralizing antibodies

Characterization of vaccine-associated enhanced respiratory disease (VAERD) in swine administered an inactivated δ-cluster influenza vaccine and challenged with pandemic A/H1N1 virus

Influenza A viruses are an important cause of respiratory disease in swine worldwide. Contemporary influenza viruses endemic in North American swine are triple reassortants with multiple subtypes displaying marked genetic and antigenic diversity. Inactivated influenza vaccines are used in swine to protect against infection and clinical disease; however, they often lack the ability to cross-protect against heterologous viruses. In addition, inactivated vaccines may enhance clinical signs and pneumonia following challenge with divergent emerging viruses that do not demonstrate serological cross-neutralizing activity. Herein we describe enhanced disease in pigs administered an inactivated H1N2 delta-cluster vaccine followed by challenge with pandemic A/H1N1 virus known as vaccine associated enhanced respiratory disease (VAERD). Pigs in the VAERD-affected group demonstrated more severe clinical signs that included coughing, respiratory distress, anorexia and lethargy in addition to elevated body temperatures compared to naive-challenged pigs. Microscopic and macroscopic pneumonia and lung lesion scores were significantly elevated in the VAERD group with increased concentrations of pulmonary pro-inflammatory cytokines. Lung lesions at 1 and 2 days post inoculation (dpi) consisted of severe necrotizing bronchitis and bronchiolitis with interlobular and alveolar edema, hemorrhage and suppurative alveolitis. Marked peribronchiolar lymphocytic cuffing and interstitial pneumonia were consistent pathological features in the lung of VAERD-affected pigs at 5 dpi. Trachea lesions in vaccinated/challenged pigs consisted of suppurative and lymphocytic tracheitis and epithelial necrosis compared to mild lesions demonstrated in non-vaccinated/challenged pigs. The immune response to the inactivated vaccine consisted of hemagglutination inhibition (HI) and serum neutralizing (SN) antibodies to homologous virus that did not cross-react with the challenge virus. In contrast, whole virus, anti-pH1N1 IgG antibodies were detected in the serum and lung of VAERD-affected pigs. However, a cross-reactive mucosal IgA response was not detected in the lung in pigs primed with the inactivated vaccine. VAERD-affected pigs demonstrated a post-challenge primary immune response consisting of anti-pH1N1 HI, SN and ELISA antibodies to the challenge virus. The results of these studies establish a consistent swine model of VAERD using an inactivated influenza vaccine followed by challenge with heterologous virus. The coincidental increase in inactivated influenza vaccine use and the evolving antigenic diversity of influenza A viruses in swine creates a realistic potential for vaccine/challenge mismatch

Quantifying the Persistence of Vaccine-Related T Cell Epitopes in Circulating Swine Influenza A Strains from 2013-2017

When swine flu vaccines and circulating influenza A virus (IAV) strains are poorly matched, vaccine-induced antibodies may not protect from infection. Highly conserved T cell epitopes may, however, have a disease-mitigating effect. The degree of T cell epitope conservation among circulating strains and vaccine strains can vary, which may also explain differences in vaccine efficacy. Here, we evaluate a previously developed conserved T cell epitope-based vaccine and determine the persistence of T cell epitope conservation over time. We used a pair-wise homology score to define the conservation between the vaccine’s swine leukocyte antigen (SLA) class I and II-restricted epitopes and T cell epitopes found in 1272 swine IAV strains sequenced between 2013 and 2017. Twenty-four of the 48 total T cell epitopes included in the epitope-based vaccine were highly conserved and found in >1000 circulating swine IAV strains over the 5-year period. In contrast, commercial swine IAV vaccines developed in 2013 exhibited a declining conservation with the circulating IAV strains over the same 5-year period. Conserved T cell epitope vaccines may be a useful adjunct for commercial swine flu vaccines and to improve protection against influenza when antibodies are not cross-reactive

Porcine Epidemic Diarrhea Virus Surface Decontamination Strategies Using Chemical Sanitizing to Reduce the Quantity of PEDV RNA on Feed Manufacturing Surfaces with Environmental Swabbing

Porcine Epidemic Diarrhea virus (PEDV) is a possible hazard in feed mills that could impact pig health. If the virus enters a feed mill, it quickly becomes widely distributed and is difficult to decontaminate from surfaces.6,7 The objective of this study was to evaluate a variety of liquid and dry chemical treatments that could be used as sanitizers to reduce the amount of PEDV found on feed manufacturing surfaces in mills. This experiment was replicated 3 times and was designed in a 5 × 10 factorial with main effects of 5 different feed manufacturing surfaces and 10 sanitizing treatments. Surfaces included stainless steel, plastic, rubber, woven polypropylene tote bag, and sealed concrete coupons (4 × 4 in). One mL (1×105 TCID50/mL) of stock PEDV was applied to each surface and allowed to dry completely for 60 min. Next, a mitigation treatment was applied for 15 min: 1) no sanitation treatment (control); 2) untreated rice hulls; 3) rice hulls treated with formaldehyde-based commercial product (Sal CURB; Kemin Inc., Des Moines, IA); 4) liquid formaldehyde-based commercial product (Sal CURB; Kemin Inc., Des Moines, IA); 5) dry commercial benzoic acid and probiotic blend (VevoVitall and CRINA; DSM Nutritional Products Inc., Parsippany, NJ); 6) liquid ammonium chloride, isopropanol, and hydrogen peroxide-based commercial food-grade sanitizer (DrySan Duo; Ecolab, St. Paul, MN); 7) liquid hydrogen peroxide commercial product (INTERvention; Virox Technologies Inc. Ontario, Canada); 8) liquid quaternary ammonium glutaraldehyde commercial product (Synergize; Preserve International, Reno NV); 9) liquid sodium hypochlorite commercial sanitizer (Bleach; Clorox, Oakland, CA); and 10) liquid medium chain fatty acid blend of caprylic, caproic, and capric acids. There were 3 replicates per treatment. The quantity of PEDV RNA was determined using qRT-PCR. All main effects, interaction, and comparisons were highly significant (P ≤ 0.001). Liquid Sal CURB and liquid bleach were the most effective chemical treatments to reduce the quantity of detectable PEDV RNA, but their application is limited due to their liquid state and potential corrosiveness. Additional research is necessary to identify the role of sanitizer on PEDV infectivity, even if RNA residue remains, and to develop dry sanitizers capable of removing PEDV RNA on swine feed manufacturing surfaces that are not corrosive

Utilizing Feed Sequencing to Decrease the Risk of Porcine Epidemic Diarrhea Virus (PEDV) Cross-contamination During Feed Manufacturing

Understanding key points of potential cross-contamination during the feed manufacturing process is important to developing efficacious methods to control or prevent transmission of pathogens into swine diets. In this study, an experiment was conducted involving 30 crossbred 10-d-old pigs that were used as a bioassay model for Porcine Epidemic Diarrhea Virus (PEDV) to determine the effects of feed batch sequencing on PEDV cross-contamination and subsequent infectivity. PEDV with a PCR cycle threshold value (Ct) of 11 was uniformly mixed into 4.5 kg of swine diet using a stainless steel bench top mixer validated for mixing efficiency. The inoculated feed was then added to 45 kg of swine diet and mixed using a 4 ft3 electric paddle mixer validated for mixing efficiency to form the positive experimental treatment. Feed was discharged, carried into a bucket elevator, and exited through a downspout. Subsequent treatment batches were formed when 50 kg of PEDV-free swine diet was sequenced immediately after the PEDV-inoculated batch without cleaning the equipment to replicate the batching process used in commercial feed mills. The subsequent sequence batches (1-4) mixed, discharged, and sampled similar to the PEDV-positive batch. Feed samples were analyzed for the presence of PEDV using PCR and bioassay. Pigs were then orally challenged with harvested supernatant. Fecal swabs were collected for PEDV PCR testing. At seven days after challenge, all pigs were necropsied. Cecum contents, ileum, and jejunum were collected for PCR, histologic, and immunohistochemistry (IHC) evaluation. Overall, the results indicate that sequencing reduced but did not eliminate the risk of PEDV transmission. All pigs (9/9) challenged with the positive treatment were infected with PEDV with feed that had a Ct mean of 31.7. The discharge for the first sequence had a Ct value of 38.1 and infected pigs were noted in pigs from one of three rooms used to bioassay the feed. The second sequence did not have detectable PEDV RNA by using PCR. Interestingly, feed from the second sequence was infectious as verified by infection in pigs from one of three rooms used for bioassay. This study is the first to demonstrate feed without detectable PEDV RNA can be infective but is similar to other research using tissue homogenates and cell culture as bioassay material. In summary, feed batch sequencing should be considered a risk mitigation strategy that can be incorporated into feed mill biosecurity programs but should not be considered a risk elimination strategy

Determining the Minimum Infectious Dose of Porcine Epidemic Diarrhea Virus (PEDV) in a Feed Matrix

Understanding the magnitude of transmissible risk Porcine Epidemic Diarrhea Virus (PEDV)-infected feed imposes and establishing the minimum infectious dose of PEDV in a feed matrix are important components in strengthening virus prevention and control methods. In this study, an experiment was performed involving 30 crossbred, 10-d-old pigs that were used as a bioassay model for the minimum infectious dose of PEDV in feed. The PEDV was first diluted using tissue culture media to form 8 serial 10-fold dilutions. An aliquot of the original stock virus at 5.6 x 105 tissue culture infectious dose/ml (TCID50/ml), each serial PEDV dilution, and one virus-negative culture medium were mixed into separate 4.5 kg batches of swine diet to form 10 experimental treatments. The feed was then subsequently evaluated for infectivity using bioassay. Fecal swabs were collected at 0, 2, 4, 6, and 7 d after challenge for PCR testing. At 7 d after challenge, all pigs were necropsied. Cecum contents, ileum and jejunum were collected for PCR, histologic and immunohistochemistry (IHC) evaluation. Overall, the results indicate 5.6 × 101 TCID50/g was the minimum PEDV dose in which infection was detected. This feed had a corresponding PCR cycle threshold (Ct) of 37. This is a relatively low dose. To illustrate, using this dose, approximately 1 g of PEDV-infected baby piglet feces could contaminate up to 500 tons of feed. The data confirm that detectable Ct values in feed can result in pig infection. Our results also illustrate that the Ct in feed that was detected as infectious can be above the detection threshold used by some diagnostic laboratories

Evaluating the Effect of Manufacturing Porcine Epidemic Diarrhea Virus (PEDV)-Contaminated Feed on Subsequent Feed Mill Environmental Surface Contamination

This study aimed to utilize the only known pilot feed mill facility approved for pathogenic feed agent use in the United States to evaluate the effect of manufacturing Porcine Epidemic Diarrhea Virus (PEDV)-contaminated feed on subsequent feed mill environmental surface contamination. In this study, PEDV inoculated feed was manufactured and conveyed on equipment along with four subsequent batches of PEDV-free feed. Equipment and environmental surfaces were sampled using swabs and analyzed for the presence of PEDV RNA by PCR. The experiment was replicated three times with decontamination of the feed mill and all equipment between replications. Overall, environmental swabs indicated widespread surface contamination of the equipment and work area after a PEDV contaminated batch of feed was processed. There was little difference in environmental sample cycle threshold (Ct) values after manufacturing each of the subsequent PEDV-negative feed batches. In summary, introduction of PEDV-infected feed into a feed mill will likely result in widespread contamination of equipment and surfaces, even after several batches of PEDV-free feed are produced. Eliminating the PEDV RNA from the feed mill environment was challenging and required procedures that are not practical to apply on a regular basis in a feed mill. This data suggests that it is extremely important to prevent the introduction of PEDV-contaminated feed, ingredients, or other vectors of transmission to minimize PEDV-risk. More research should be conducted to determine if contaminated surfaces can lead to PEDV infectivity and to determine the best feed mill PEDV-decontamination strategies