Master of Science / Grain Science and Industry / Cassandra K. Jones / Feed mill biosecurity is a growing concern for the feed industries, especially since the entry of Porcine Epidemic Diarrhea Virus (PEDV) to the United States. Porcine Epidemic Diarrhea Virus (PEDV) is primarily transmitted by fecal-oral contamination. However, research has confirmed swine feed and ingredients as potential vectors of transmission, so strategies are needed to mitigate PEDV in feed. The objective of the first experiment was to evaluate the effectiveness of various chemical additives to prevent or mitigate PEDV in swine feed and ingredients that had been contaminated post-processing. Time, formaldehyde, medium chain fatty acids, essential oils, and organic acids all enhance the degradation of PEDV RNA in swine feed and ingredients, but their effectiveness varies within matrix. Notably, the medium chain fatty acids were equally as successful at mitigating PEDV as a commercially-available formaldehyde product. Salmonella is also another potential feed safety hazard in animal feed ingredients. Thermal mitigation of Salmonella in ingredients and feed manufacturing is effective, but it does not eliminate the potential for cross contamination. Therefore, the objective of the second experiment was to evaluate the effectiveness of chemicals to mitigate Salmonella cross-contamination in rendered proteins over time. Both chemical treatment and time reduced Salmonella concentrations, but their effectiveness was again matrix dependent. Chemical treatment with medium chain fatty acids or a commercial formaldehyde product was most effective at mitigating Salmonella in rendered protein meals. The final experiment was conducted to evaluate the effectiveness of a dry granular acid, sodium bisulfate (SBS; Jones-Hamilton, Co., Waldridge, OH), to mitigate contamination of Salmonella in poultry feed. A surrogate organism, Enterococcus faecium, was utilized for this research in order to evaluate the effectiveness of SBS. Thermal processing, SBS concentration, and time all impacted biological pathogen levels in poultry diets, and including a dry granular acid may be an effective method to reduce pathogen risk. However, the most significant reduction of Enterococcus faecium was due to thermal mitigation. Notably, pelleting reduced Enterococcus faecium by 2-3 logs on day 0. In summary, both thermal processing and chemical inclusion can be used to reduce the risk of microbial pathogens in feed.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Daniel U. Thomson / A series of studies were conducted in order to develop, test, implement, and utilize an objective and comprehensive gross pathology scoring system for cattle at slaughter. Individual lung, liver, and rumen gross pathology data was collected from 19,229 head of cattle and corresponding individual pre-harvest and carcass data for a subset of 13,226 head.. Across the entire population 22.6% and 9.8% of cattle displayed mild and severe lesions, respectively. Severe lung lesions at the time of slaughter were associated with a decreased ADG of 0.07 kg/ day and a carcass weight 7.1 kg less than that of their cohorts with no visible signs of pulmonary BRDC lesions (P < 0.01). Overall, 68.6 % of cattle observed had normal livers, free from abscesses and other abnormalities. Cattle with a severe liver abscess at the time of slaughter were associated with a 0.10 kg/day during the feeding period (P < 0.01). Of cattle severely affected by liver abscesses (A+, 4.6%), 14.9% also displayed severe BRDC lung lesions and 28.3 % of cattle displayed mild BRDC lung lesions. Rumenitis lesions were observed in 24.1% of the overall study population. Severe rumenitis lesions were associated with a significant decrease in average daily gain and carcass weight (0.03kg/day and 2.20 kg, respectively, P < 0.01). The system was also implemented on a population of cull cows at a commercial abattoir in the Great Lakes region of the U.S. (n=1,461; 87% Holstein, 13% other cows). Severe liver abscesses, were observed in 18.5% of cull cows at slaughter. Severe rumenitis lesions or rumenitis scars were observed in 10% and severe BRDC lesions were observed in 10.3% o of the population. A prospective study of a commercially available, direct fed microbial oral drench of Megasphaera elsdenii (NCIMB 41125) was conducted in 4,863 head of yearling feeder cattle. No significant effects of treatment were detected for final live weight (599 vs. 601 kg; P=0.79) or hot carcass weight (386 vs. 387 kg P=0.81) for Con and M.e., respectively. Fourteen point two percent and 14.0% of Con and M.e., respectively displayed a liver abscess of varying severity at the time of slaughter. Overall, 8.27 and 7.96% % of Con and M.e. cattle were observed with an altered rumen epithelial health status. The ordinal odds ratio of a M.e. treated animal having a more severe liver abscess score or rumen health score was not significant (Estimate: 0.96, 95% C.L. 0.733-1.259, P=0.771; Estimate: 1.01, 95% C.L. 0.625-1.63 P=0.96, respectively.) Comprehensive monitoring of gross pathology at slaughter is commercially plausible and provides valuable data for veterinarians, nutritionists and management personnel.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / With introduction of the 2009 pandemic H1N1 virus (pH1N1) into swine herds, reassortment between the pH1N1 and endemic swine influenza viruses (SIVs) has been reported worldwide. Recently, reassortant H3N2 and H1N2 variant SIVs that contain the M gene from pH1N1 virus and the remaining seven genes from North American triple-reassortant (TR) SIVs have emerged. These variant viruses have caused more than 300 cases of human infections and one death in the USA, creating a major public health concern. To date, the pathogenicity and transmissibility of H1N2 variant viruses in pigs has not been investigated. Through passive surveillance, we have isolated two genotypes of reassortant H1N2 viruses with pH1N1 genes from diseased pigs in Kansas. Full genome sequence and phylogenetic analysis showed that one is a swine H1N2 variant virus (swH1N2v) with the M gene from pH1N1; the other is a reassortant H1N2 virus (2+6 rH1N2) with six internal genes from pH1N1 and the two surface genes from endemic North American TR H1N2 SIVs. Furthermore, we determined the pathogenicity and transmissibility of the swH1N2v, a human H1N2 variant (huH1N2v), and the 2+6 rH1N2 in pigs using an endemic TR H1N2 SIV (eH1N2) isolated in 2011 as a control. All four viruses were able to infect pigs and replicate in the lungs. Both H1N2 variant viruses caused more severe lung lesions in infected pigs when compared to the eH1N2 and 2+6 rH1N2 viruses. Although all four viruses are transmissible in pigs and were detected in the lungs of contact animals, the swH1N2v shed more efficiently than the other three viruses in the respective sentinel animals. The huH1N2v displayed delayed and inefficient nasal shedding in sentinel animals. Taken together, the human and swine H1N2 variant viruses are more pathogenic and the swH1N2v more transmissible in pigs and could pose a threat to public and animal health.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ying Fang / Porcine reproductive and respiratory syndrome virus (PRRSV) employs multiple functions to modulate host’s innate immune response, and several viral nonstructural proteins (nsps) are major players. In this dissertation, the research was mainly focused on identification and functional dissection of ORF1a-encoded nsps. PRRSV replicase polyproteins encoded by ORF1a region are predicted to be processed into at least ten nonstructural proteins. In chapter 2, these predictions were verified by using a panel of newly established antibodies specific to ORF1a-encoded nsps. Most predicted nsps (nsp1β, nsp2, nsp4, nsp7α, nsp7β and nsp8) were identified, and observed to be co-localized with de novo-synthesized viral RNA in the perinuclear region of the cell. Among all PRRSV proteins screened, nsp1β is the strongest type I interferon antagonist. In chapter 3, mutagenesis analysis of nsp1β was performed to knock down nsp1β’s IFN antagonist function. A highly conserved motif, GKYLQRRLQ, was determined to be critical for nsp1β’s ability to suppress IFN-β and reporter gene expression. Double mutations introduced in this motif, K130A/R134A (type 1 PRRSV) or K124A/R128A (type 2 PRRSV), improved PRRSV’s ability to stimulate the expression of IFN-α, IFN-β and ISG15. In addition to its critical roles involving in modulating host innate immune response, in the studies of Chapter 4, we demonstrated that PRRSV nsp1β functions as a transactivator to induce the -2/-1 ribosomal frameshifting in nsp2, which results in expression of two novel PRRSV proteins, nsp2TF and nsp2N. The conserved motif GKYLQRRLQ is also determined to be critical for the transactivation function of nsp1β. In chapter 5, the interferon antagonist, de-Ub and de-ISGylation activity of newly identified nsp2TF and nsp2N were evaluated. In vitro and in vivo characterization of three nsp2TF-deficient recombinant viruses indicated that all mutant viruses have improved ability to stimulate the innate immune response and provide improved protection in mutant virus-vaccinated animals. In summary, this study verified the previously predicted PRRSV pp1a processing products, further evaluated the function of nsp1β and nsp2-related proteins. These data obtained here will provide basic knowledge for future development of vaccines and control measurements.