Drew R. Magstadt

Pathogenesis of porcine epidemic diarrhea virus isolate (US/Iowa/18984/2013) in 3-week-old weaned pigs

Porcine epidemic diarrhea virus (PEDV) is associated with clinical diarrhea in naïve swine of all ages. This report describes timing of antibody generation and disease progression following infection with a US PEDV isolate by assessing fecal viral shedding, morphometric analysis of intestinal lesions, and magnitude of immunohistochemical staining. Sixty-three, 3-week-old pigs were randomly allocated into control (n=27) and challenged (n=36) groups. Challenged pigs were administered 1 mL of 1 × 10(3) PFU/mL of US/Iowa/18984/2013 PEDV isolate by oro-gastric gavage. Three control and four challenged pigs were necropsied on days post-inoculation (dpi) 1, 2, 3, 4, 7, and weekly thereafter, until study termination on dpi 35. Clinical disease, fecal shedding, body weight, and temperature were monitored during the study period. Diarrhea was observed in challenged pigs beginning for some on dpi 2, affecting a majority of pigs by dpi 6 and subsiding by dpi 10. Average daily gain was significantly lower (P<0.001) for one week post-infection in challenged pigs. PEDV was detected in feces by PCR on dpi 1 and continued in a subset of pigs until dpi 24. PEDV-specific antigen was detected in villous enterocytes of challenged pigs by immunohistochemistry (IHC) on dpi 1, 2, 3, 4, 7, and 14. Microscopic lesions included severe diffuse atrophic enteritis with significantly reduced (P<0.001) villous length observed on dpi 3, 4, and 7. Under the conditions of this study, fecal shedding of PEDV and IHC staining can precede and continue beyond the observation of clinical signs, thus increasing the risk of viral transmission.

Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets

Abstract Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2009–2010 and reported in United States swine for the first time in February 2014. However, diagnostic tools other than polymerase chain reaction for PDCoV detection were lacking and Koch׳s postulates had not been fulfilled to confirm the pathogenic potential of PDCoV. In the present study, PDCoV peptide-specific rabbit antisera were developed and used in immunofluorescence and immunohistochemistry assays to assist PDCoV diagnostics. The pathogenicity and pathogenesis of PDCoV was investigated following orogastric inoculation of 5-day-old piglets with a plaque-purified PDCoV cell culture isolate (3×104 TCID50 per pig). The PDCoV-inoculated piglets developed mild to moderate diarrhea, shed increasing amount of virus in rectal swabs from 2 to 7 days post inoculation, and developed macroscopic and microscopic lesions in small intestines with viral antigen confirmed by immunohistochemistry staining. This study experimentally confirmed PDCoV pathogenicity and characterized PDCoV pathogenesis in neonatal piglets.

Effect of porcine epidemic diarrhea virus infectious doses on infection outcomes in naïve conventional neonatal and weaned pigs

Porcine epidemic diarrhea virus (PEDV) was identified in the United States (U.S.) swine population for the first time in April 2013 and rapidly spread nationwide. However, no information has been published regarding the minimum infectious dose (MID) of PEDV in different pig models. The main objective of this study was to determine the oral minimum infectious dose of PEDV in naïve conventional neonatal piglets and weaned pigs. A U.S. virulent PEDV prototype isolate (USA/IN19338/2013) with known infectious titer was serially ten-fold diluted in virus-negative cell culture medium. Dilutions with theoretical infectious titers from 560 to 0.0056 TCID50/ml together with a medium control were orogastrically inoculated (10ml/pig) into 7 groups of 5-day-old neonatal pigs (n = 4 per group) and 7 groups of 21-day-old weaned pigs (n = 6 per group). In 5-day-old pigs, 10ml of inoculum having titers 560–0.056 TCID50/ml, corresponding to polymerase chain reaction (PCR) cycle threshold (Ct) values 24.2–37.6, resulted in 100% infection in each group; 10ml of inoculum with titer 0.0056 TCID50/ml (Ct>45) caused infection in 25% of the inoculated pigs. In 21-day-old pigs, 10ml of inoculum with titers 560–5.6 TCID50/ml (Ct 24.2–31.4) resulted in 100% infection in each group while 10ml of inoculum with titers 0.56–0.0056 TCID50/ml (Ct values 35.3 –>45) did not establish infection in any pigs under study conditions as determined by clinical signs, PCR, histopathology, immunohistochemistry, and antibody response. These data reveal that PEDV infectious dose is age-dependent with a significantly lower MID for neonatal pigs compared to weaned pigs. This information should be taken into consideration when interpreting clinical relevance of PEDV PCR results and when designing a PEDV bioassay model. The observation of such a low MID in neonates also emphasizes the importance of strict biosecurity and thorough cleaning/disinfection on sow farms.

Identification of a Divergent Lineage Porcine Pestivirus in Nursing Piglets with Congenital Tremors and Reproduction of Disease following Experimental Inoculation

Congenital tremors is a sporadic disease of neonatal pigs characterized by action-related repetitive myoclonus. A majority of outbreaks of congenital tremors have been attributed to an unidentified virus. The objectives of this project were to 1) detect potential pathogen(s) in samples from piglets with congenital tremors and 2) develop an infection model to reproduce disease. Using next-generation sequencing, a divergent lineage pestivirus was detected in piglets with congenital tremors. The virus was originally most closely related to a bat pestivirus but is now more closely related to a recently published novel porcine pestivirus provisionally named atypical porcine pestivirus. A quantitative real-time PCR detected the virus in samples from neonatal piglets with congenital tremors from two separate farms, but not in samples from unaffected piglets from the same farm. To fulfill the second objective, pregnant sows were inoculated with either serum containing the pestivirus or PBS (control) by intravenous and intranasal routes simultaneously with direct inoculation of fetal amniotic vesicles by ultrasound-guided surgical technique. Inoculations were performed at either 45 or 62 days of gestation. All sows inoculated with the novel pestivirus farrowed piglets affected with congenital tremors while PBS-inoculated control piglets were unaffected. Tremor severity for each piglet was scored from videos taken 0, 1 and 2 days post-farrowing. Tremor severity remained relatively constant from 0 to 2 days post-farrowing for a majority of piglets. The prevalence of congenital tremors in pestivirus-inoculated litters ranged from 57% (4 out of 7 affected piglets) to 100% (10 out of 10 affected piglets). The virus was consistently detected by PCR in tissues from piglets with congenital tremors but was not detected in control piglets. Samples positive by PCR in greater than 90% of piglets sampled included brainstem (37 out of 41), mesenteric lymph node (37 out of 41), tracheobronchial lymph node (37 out of 41), and whole blood (19 out of 20). Although the first description of congenital tremors was in 1922, this is the first reported reproduction of congenital tremors following experimental inoculation with a divergent lineage porcine pestivirus. Studies investigating disease mechanism, epidemiology, and diagnostic assay development are needed to better understand the pathophysiology of congenital tremors due to this pestivirus.

Characterization of Porcine Epidemic Diarrhea Virus Isolate US/Iowa/18984/2013 Infection in 1-Day-Old Cesarean-Derived Colostrum-Deprived Piglets

Porcine epidemic diarrhea virus (PEDV) was first recognized in North America in April 2013 and has since caused devastating disease. The objective of this study was to characterize disease and viral detection associated with an original North American PEDV isolate inoculated in neonatal piglets. Thirty-six 1-day-old cesarean-derived and colostrum-deprived piglets were randomly assigned to the control (n = 16) or challenged group (n = 20); the latter were orogastrically inoculated with 1 ml of US/Iowa/18984/2013 PEDV isolate titered at 1 × 103 plaque-forming units per milliliter. Rectal swabs were collected from all piglets prior to inoculation and every 12 hours postinoculation (hpi) thereafter, with 4 control and 5 challenged piglets euthanized at 12, 24, 48, and 72 hpi. One piglet had a positive real-time quantitative polymerase chain reaction test on rectal swab at 12 hpi, and all remaining piglets were positive thereafter, with highest viral quantities detected at 24 and 36 hpi. Diarrhea was evident in 30% and 100% of challenged piglets at 18 and 24 hpi, respectively. Viral antigen was detected in enterocytes by immunohistochemistry in the duodenum and ileum of piglets euthanized at 12 hpi and was apparent throughout the small intestine of all piglets thereafter, with villus height:crypt depth ratios consistently below 4:1. Viremia was confirmed in 18 of 20 pigs at euthanasia. Clinical disease was severe and developed rapidly following infection with an original North American PEDV isolate, with lesions, viremia, and antigen detection possible by 12 hpi.

Investigation of the Impact of Increased Dietary Insoluble Fiber through the Feeding of Distillers Dried Grains with Solubles (DDGS) on the Incidence and Severity of Brachyspira-Associated Colitis in Pigs

Diet has been implicated as a major factor impacting clinical disease expression of swine dysentery and Brachyspira hyodysenteriae colonization. However, the impact of diet on novel pathogenic strongly beta-hemolytic Brachyspira spp. including “B. hampsonii” has yet to be investigated. In recent years, distillers dried grains with solubles (DDGS), a source of insoluble dietary fiber, has been increasingly included in diets of swine. A randomized complete block experiment was used to examine the effect of increased dietary fiber through the feeding of DDGS on the incidence of Brachyspira-associated colitis in pigs. One hundred 4-week-old pigs were divided into five groups based upon inocula (negative control, Brachyspira intermedia, Brachyspira pilosicoli, B. hyodysenteriae or “B. hampsonii”) and fed one of two diets containing no (diet 1) or 30% (diet 2) DDGS. The average days to first positive culture and days post inoculation to the onset of clinical dysentery in the B. hyodysenteriae groups was significantly shorter for diet 2 when compared to diet 1 (P = 0.04 and P = 0.0009, respectively). A similar difference in the average days to first positive culture and days post inoculation to the onset of clinical dysentery was found when comparing the “B. hampsonii” groups. In this study, pigs receiving 30% DDGS shed on average one day prior to and developed swine dysentery nearly twice as fast as pigs receiving 0% DDGS. Accordingly, these data suggest a reduction in insoluble fiber through reducing or eliminating DDGS in swine rations should be considered an integral part of any effective disease elimination strategy for swine dysentery.

Effect of pelleting on survival of porcine epidemic diarrhea virus-contaminated feed

Abstract Porcine epidemic diarrhea virus (PEDV) is a heat-sensitive virus that has devastated the U.S. swine industry. Because of its heat sensitivity, we hypothesized that a steam conditioner and pellet mill mimicking traditional commercial thermal processing may mitigate PEDV infectivity. Pelleting, a common feed processing method, includes the use of steam and shear forces, resulting in increased temperature of the processed feed. Two thermal processing experiments were designed to determine if different pellet mill conditioner retention times and temperatures would impact PEDV quantity and infectivity by analysis of quantitative reverse transcription PCR and bioassay. In Exp. 1, a 3 · 3 · 2 factorial design was used with 3 pelleting temperatures (68.3, 79.4, and 90.6°C), 3 conditioning times (45, 90, or 180 s), and 2 doses of viral inoculation (low, 1 · 102 tissue culture infectious dose50 (the concentration used to see cytopathic effect in 50% of the cells)/g, or high, 1 · 104 tissue culture infectious dose50/g). Noninoculated and PEDV-inoculated unprocessed mash were used as controls. The low-dose PEDV–infected mash had 6.8 ± 1.8 cycle threshold (Ct) greater (P < 0.05) PEDV than the high-dose mash. Regardless of time or temperature, pelleting reduced (P < 0.05) the quantity of detectable viral PEDV RNA compared with the PEDV-inoculated unprocessed mash. Fecal swabs from pigs inoculated with the PEDV-positive unprocessed mash, regardless of dose, were clinically PEDV positive from 2 to 7 d (end of the trial) after inoculation. However, if either PEDV dose of inoculated feed was pelleted at any of the 9 tested conditioning time · temperature combinations, no PEDV RNA was detected in fecal swabs or cecum content. Based on Exp. 1 results, a second experiment was developed to determine the impact of lower processing temperatures on PEDV quantity and infectivity. In Exp. 2, PEDV-inoculated feed was pelleted at 1 of 5 conditioning temperatures (37.8, 46.1, 54.4, 62.8, and 71.1°C) for 30 s. The 5 increasing processing temperatures led to feed with respective mean Ct values of 32.5, 34.6, 37.0, 36.5, and 36.7, respectively. All samples had detectable PEDV RNA. However, infectivity was detected by bioassay only in pigs from the 37.8 and 46.1°C conditioning temperatures. Experiment 2 results suggest conditioning and pelleting temperatures above 54.4°C could be effective in reducing the quantity and infectivity of PEDV in swine feed. However, additional research is needed to prevent subsequent recontamination after pelleting as it is a point-in-time mitigation step.

Toxocara vitulorum infection in a cohort of beef calves in Iowa

Toxocara vitulorum, an ascarid that infects bovines, is largely considered a parasite of tropical and sub-tropical areas of the world. Infections in more temperate climates have been reported but little is known about T. vitulorum transmission in North America. Herein, we detail an investigation of an Iowa beef farm where necropsy of a 20 day old beef calf revealed a mass of large ascarid nematodes occluding the small intestine. Gross histopathological, and molecular features of nematodes recovered from the calf were consistent with T. vitulorum. Fecal samples obtained from calves in the herd revealed 14 of 34 (40%) were shedding T. vitulorum eggs. Calves continued to shed eggs following a single dose of a topical moxidectin product. These results suggest that T. vitulorum may be more widespread in the United States than previously recognized. Due to the pathogenic nature of this parasite, further investigation into the prevalence and transmission of T. vitulorum in North America is warranted.

Outbreak of H5N2 highly pathogenic avian Influenza A virus infection in two commercial layer facilities lesions and viral antigen distribution

The largest outbreak of highly pathogenic avian Influenza A virus (HPAIV) infection in U.S. history began in December 2014 resulting in the euthanasia of millions of birds and collateral economic consequences to the U.S. poultry industry. We describe 2 cases of H5N2 HPAIV infection in laying hens in Iowa. Following a sharp increase in mortality with minimal clinical signs, 15 dead birds, from 2 unrelated farms, were submitted to the Iowa State University Veterinary Diagnostic Laboratory. Common lesions included diffuse edema and multifocal hemorrhage of the comb, catarrhal exudate in the oropharynx, and multifocal tracheal hemorrhage. Less common lesions included epicardial petechiae, splenic hemorrhage, and pancreatic necrosis. Influenza A virus nucleoprotein was detected by immunohistochemistry in multiple cell types including ependymal cells, the choroid plexus, neurons, respiratory epithelium and macrophages in the lung, cardiac myocytes, endothelial cells, necrotic foci in the spleen, Kupffer cells in the liver, and necrotic acinar cells in the pancreas. Real-time polymerase chain reaction and sequencing confirmed H5N2 HPAIV with molecular characteristics similar to other contemporary U.S. H5N2 HPAIVs in both cases.

Treatment history and antimicrobial susceptibility results for Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni isolates from bovine respiratory disease cases submitted to the Iowa State University Veterinary Diagnostic Laboratory from 2013 to 2015:

Bovine respiratory disease is the most costly disease facing the cattle industry. Increasing resistance to antimicrobial treatment has been presented as a significant contributing factor, often through summarized susceptibility testing data. We assessed the relationship between previous antimicrobial treatment and antimicrobial susceptibility results from isolates of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni cultured from bovine respiratory cases submitted to the Iowa State University Veterinary Diagnostic Laboratory from 2013 to 2015. Antimicrobial susceptibility data from 1,251 bacterial isolates were included for analysis. More bacterial isolates from cattle that received antimicrobial treatment showed resistance compared to isolates from untreated cattle, and the percentage of resistant isolates increased as the number of antimicrobial treatments increased. Resistance to enrofloxacin, spectinomycin, tilmicosin, and tulathromycin was present in >75% of M. haemolytica isolates from cattle that had received 3 or more antimicrobial treatments; resistance to each of those 4 antimicrobials was present in ≤10% of M. haemolytica isolates from untreated cattle. Similar but less dramatic trends were apparent for isolates of P. multocida and H. somni. The percentage of multi-drug resistant bacterial isolates also increased with the number of treatments. Results of our study suggest that previous antimicrobial treatment may have a profound effect on antimicrobial susceptibility testing. Summarized susceptibility results from diagnostic laboratories should not be used to make generalized statements regarding trends in antimicrobial resistance without providing context regarding antimicrobial treatment history.