Dissertations & Theses in Veterinary and Biomedical Science Copyright (c) 2013 University of Nebraska - Lincoln All rights reserved. http://digitalcommons.unl.edu/vetscidiss Recent documents in Dissertations & Theses in Veterinary and Biomedical Science en-us Thu, 24 Jan 2013 23:42:52 PST 3600 STRUCTURE-FUNCTION ANALYSIS OF A PROTEIN ENCODED BY THE BHV-1 LATENCY RELATED GENE http://digitalcommons.unl.edu/vetscidiss/13 http://digitalcommons.unl.edu/vetscidiss/13 Thu, 29 Nov 2012 11:50:19 PST Bovine herpes virus 1 (BHV-1) is a significant viral pathogen in cattle that induces a myriad of clinical symptoms. These symptoms include: conjunctivitis, upper respiratory tract infections, genital disorders, and abortions. BHV-1 infection can also lead to transient immune-suppression, which predisposes cattle to secondary bacterial infection leading to life-threatening pneumonia referred to as bovine respiratory disease (BRD). Following acute infection, BHV-1 establishes latency in sensory neurons within trigeminal ganglia. Reactivation of the virus can occur periodically, resulting in virus transmission. The latency-related (LR) RNA is the only abundantly expressed transcript in latently infected sensory neurons and it encodes several proteins, including ORF2, as well as two micro-RNAs. My dissertation work has focused on trying to understand how the LR gene is able to promote establishment and maintenance of latency, specifically through elucidating the mechanism of function of the LR encoded protein ORF2. ORF2 inhibits apoptosis and also interacts with Notch signaling receptors, inhibiting their ability to activate certain BHV-1 promoters and enhance productive infection. The studies presented here identified, through mutational analysis, distinct domains in ORF2 that regulate its stability, localization, and functional properties. Furthermore, ORF2 is able to affect different cellular processes and promote a mature neuronal phenotype through inhibition of the Notch pathway. Lastly, a subset of ORF2 was associated with


chromatin and preferentially associated with single stranded DNA. Collectively, these studies suggest that ORF2 by interfering with apoptosis and the Notch pathway and through its ability to bind DNA plays a role in regulating certain aspects of the latency-reactivation cycle. ORF2, in general, enhances survival of infected neurons, promotes a mature neuronal phenotype and consequently increases the pool of latently infected neurons.

Adviser: Clinton Jones

]]> Devis Sinani Expression and Distribution of Thiol- regulating Enzyme, Glutaredoxin 2 in Porcine Ocular Tissues http://digitalcommons.unl.edu/vetscidiss/12 http://digitalcommons.unl.edu/vetscidiss/12 Thu, 26 Jul 2012 06:24:57 PDT Glutaredoxin 2 (Grx2), a thiol-regulating enzyme of oxidoreductase family and a mitochondrial isozyme of glutaredoxin 1, was discovered 11 years ago in our laboratory. Grx2 is present in the lens where it shows dethiolase, peroxidase, and ascorbate recycling activities. Recently, Grx2 has also been identified to protect the mitochondrial electron transport system with anti-apoptotic function. Since other eye tissues besides the lens are rich in mitochondria and are very sensitive to oxidative stress, we speculate the presence of Grx2 therein as an important redox regulator. This study is to investigate the expression and distribution of Grx2 in ocular tissues using porcine eye as a model.

Fresh enucleated porcine eyes obtained from a local abattoir were immediately dissected into cornea, iris, the lens, vitreous humor, ciliary body, retina, and optic nerve; frozen in dry ice; and stored at -80oC. Each sample with 3 tissues pooled was homogenized in 1.0 ml of ice-cold 10 mM HEPES buffer (pH 7.2) containing 225 mM D-mannitol, 65 mM sucrose and 1mM EGTA in a glass-to-glass homogenizer, followed by a series of centrifugations to remove tissue debris while isolating mitochondrial fraction. Mitochondrial sample proteins of each sample were separated by 12% SDS-PAGE, followed by Western blot analysis to detect Grx2 by a specific Grx2 antibody. The same tissue sample with 5mg/ml mitochondrial protein was completely dissolved in 1% lauryl maltoside, and measured for Grx2 enzyme activity following the published procedure.

Western blots showed the expression of Grx2 in all the tested ocular tissues, except vitreous humor. Relative expression of Grx2 to the positive control, mouse mitochondrial liver homogenate, revealed that the ciliary body had the highest expression ratio (26.64), followed by the retina (11.92), and optic nerve (8.60). The lens had the lowest expression ratio of 0.75, while the vitreous humor did not show any Grx2 positive band. Enzyme activity assays showed that the retina had the highest Grx2 specific activity (3.89 mU/mg protein), closely followed by ciliary body (3.10 mU/mg), lens (0.58 mU/mg), and optic nerve (0.32 mU/mg). Vitreous humor had no Grx2 activity.

In conclusion, Grx2 was found in all porcine ocular tissues except for vitreous humor. The Grx2 protein expression level was higher in eye tissues rich in mitochondria (ciliary body and retina), corroborating with their high Grx2 activity. The rich presence of Grx2 in these tissues is consistent with their known sensitivity to oxidative stress.

Advisor: Marjorie F. Lou

]]> Bijaya Prasad Upadhyaya Cell-Mediated Immunity in Porcine Reproductive and Respiratory Syndrome Virus http://digitalcommons.unl.edu/vetscidiss/11 http://digitalcommons.unl.edu/vetscidiss/11 Thu, 17 May 2012 14:47:40 PDT Porcine reproductive and respiratory syndrome virus is a significant swine pathogen which exhibits considerable sequence diversity. In an attempt to identify highly conserved T-cell epitopes contained in proteins of this virus, we examined heptadecamer peptides spanning the sequence of the PRRSV nonstructural proteins 9, 10 and 11, all of them are highly conserved, for their ability to elicit a recall proliferative and interferon-gamma response in peripheral blood mononuclear cells obtained from pigs immunized against the type-II PRRSV strain FL-12. These studies led to the identification of seven peptides, two from each NSP 9 and NSP 10 and, three from NSP 11 that appear to contain T-cell epitopes. Comparison of the amino acid sequence of these seven peptide sequences to the analogous sequences from a diverse sample of type-II PRRSV strains indicated that these sequences are highly conserved and thus contain highly conserved T-cell epitopes. The identified epitopes may be important in the formulation of immunogens to provide broad cross-protection against diverse PRRSV strains.

Advisor: Fernando A. Osorio

]]> Rajeshwari Parida MODELING THE EFFICACY AND EFFECTIVENESS OF ESCHERICHIA COLI O157:H7 PRE-HARVEST INTERVENTIONS http://digitalcommons.unl.edu/vetscidiss/10 http://digitalcommons.unl.edu/vetscidiss/10 Mon, 23 Apr 2012 09:17:17 PDT Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) is one of the leading causes of hemolytic uremic syndrome in humans. Reducing the prevalence of STEC O157 in live cattle may reduce ground beef prevalence and subsequent human illness. Type III secreted protein vaccines (TTSP) reduce fecal shedding of STEC O157 in cattle. However, pre-harvest vaccines have yet to be adopted by the beef industry. The objectives of this thesis were to 1) conduct a meta-analysis to test factors effecting efficacy of a 3-dose regimen TTSP vaccine product, and 2) use stochastic simulation to model the usefulness of preharvest control measures. In the meta-analysis, data was used from four randomized controlled vaccine trials conducted from 2002-2008 at the University of Nebraska–Lincoln (n=184 pens, 1,462 cattle). Results indicated, study, challenge load, and days from administration of the last dose of the vaccine did not modify the measure of vaccine efficacy for a 3-dose regimen TTSP vaccine product. Model adjusted efficacy was 48% (95%CI, 0.37-0.57). In the modeling study, we simulated the pen-level fecal shedding prevalence distribution of cattle fed in the summertime and vaccinated with a TTSP vaccine and compared it to the winter fecal shedding prevalence distribution. Model inputs were previously observed frequency distributions for number of animals within a pen, and pen-level fecal shedding prevalence for summer and winter. Uncertainty about vaccine efficacy was modeled as a log-normal distribution (μ=58, SE=0.1393). The simulation was performed 5,000 times. Vaccination with a TTSP vaccine reduced summertime pen prevalence distributions of STEC O157 to levels comparable to wintertime pen prevalence, with the major effect being reduced variability in fecal shedding prevalence. Our simulation model should be a useful tool for food safety decision makers evaluating the usefulness of pre-harvest interventions.

]]> Amanda R. Vogstad ROLE OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS NON-STRUCTURAL PROTEIN 1 IN VIRAL REPLICATION AND PATHOGENESIS http://digitalcommons.unl.edu/vetscidiss/9 http://digitalcommons.unl.edu/vetscidiss/9 Wed, 27 Jul 2011 06:40:36 PDT Porcine reproductive and respiratory syndrome virus (PRRSV) is an infectious agent of significant concern to the global swine industry. PRRSV infection of pigs is initiated by a long viremia after which the virus enters in an extended persistent phase of 3-4 months that ultimately is resolved by the immune system. The delayed and weak host adaptive response is responsible for the protracted convalescence period. An initial sub-optimal innate immune response is postulated to be the reason behind such meager adaptive immune response. A major focus of the studies undertaken in this dissertation was to identify the viral non-structural proteins (nsps) involved in antagonizing cellular interferon (IFN) production, a principal component of host innate defense system. Among the four different nsps identified from initial cursory screening, nsp1 exhibited the strongest suppression of IFN induction. Nsp1α and nsp1β, the proteolytic products of nsp1 were both found to down-regulate dsRNA-induced activation of interferon regulatory factor 3 (IRF3). Furthermore, Nsp1β specifically antagonized IRF3 phosphorylation and nuclear translocation. In order to determine the amino acid residues responsible for the anti-IFN property of nsp1α and nsp1β, we prepared alanine-scanning mutants of both proteins. These mutants were probed for their ability to alleviate the IFN-suppression in reporter assays. Several candidates with variable degrees of relief were identified. A recombinant PRRSV with the respective mutations incorporated in nsp1β induced higher level of type I IFN and was attenuated in vitro. However, in infected swine the mutant virus quickly reverted to acquire the wild type IFN-suppression phenotype. To further understand the role of nsp1β in PRRSV life cycle, we employed co-immunoprecipitation coupled with mass spectrometry analysis to identify host cellular factors that interact with nsp1β. Here, we characterized nsp1β’s interaction with cellular poly(C) binding protein (PCBP)-1 and 2. Both PCBP1 and PCBP2 associated with several components of viral replication and transcription apparatus. Using various biochemical assays, I demonstrated that both PCBP1 and PCBP2 are important for transcription and/or replication of the viral genome.

Adviser: Fernando Osorio

]]> Lalit Beura IDENTIFICATION OF HOST PROTEINS REQUIRED FOR VESICULAR STOMATITIS VIRUS INFECTION http://digitalcommons.unl.edu/vetscidiss/8 http://digitalcommons.unl.edu/vetscidiss/8 Mon, 25 Jul 2011 08:58:59 PDT Viruses usurp host cell pathways for different stages of their infection. Understanding virus-host interaction will be invaluable to elucidate molecular mechanisms of virus infection and to identify drug targets. In order to identify such critical cellular genes in vesicular stomatitis virus (VSV, a model non-segmented negative strand RNA virus) infection, we developed a stable cell line constitutively expressing replication proteins of VSV. Attempts to establish a cell line replicating a sub-genomic replicon was not successful because of induction of interferon response by replication of viral genomic analog. Subsequently, we used siRNA technology and conducted a genome-wide siRNA screen in HeLa cells to identify host factors regulating VSV infection. A total of 23,000 human genes were knocked down individually, and their effect on viral infection was interrogated using a high-throughput cell-based assay. Our study identified several previously unknown host proteins required for VSV infection. Bioinformatics analysis predicted enrichment of several biological functions among these proteins and some of them are commonly utilized by other pathogens such as human immune deficiency virus (HIV), hepatitis C virus (HCV) and Influenza virus. We also noted that 35% of these genes (25 out of 72) are required for lymphocytic choriomeningitis virus (LCMV) and human parainfluenza virus type 3 (HPIV3) infection, suggesting evolutionary conserved mechanisms of virus-host interactions. Further studies focusing on host coatomer complex 1 (COPI) identified a role of COPI in early stage of VSV infection. The effect of COPI is mediated at the level of viral RNA synthesis. COPI functions are required not only for VSV but also for LCMV and HPIV. ADP ribosylation factor 1 (ARF1), the immediate upstream modulator of COPI was found as a required factor for VSV RNA synthesis. ARF1 is activated by the Golgi-associated brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1) which was found to be a critical determinant of VSV RNA synthesis. These studies suggested that the components of the cellular secretory pathway are required for VSV RNA synthesis.

Adviser: Asit K. Pattnaik

]]> Debasis Panda EFFECT OF THE INFECTION WITH PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS ON THE REGULATION OF CYTOKINES - TUMOR NECROSIS FACTOR-ALPHA AND INTERLEUKIN-10 http://digitalcommons.unl.edu/vetscidiss/7 http://digitalcommons.unl.edu/vetscidiss/7 Thu, 14 Jul 2011 09:18:41 PDT Porcine reproductive and respiratory syndrome virus (PRRSV) causes late-term abortion in sows and pneumonia in growing piglets. PRRSV evades the host immune response by several mechanisms, including the modulation of cytokine secretions in infected pigs, which is the subject of this dissertation. Particularly, PRRSV reduces the secretion of the pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α) but increases the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). The latter effect, however, is PRRSV strain-specific. In this dissertation, we have examined mechanisms by which PRRSV regulates TNF-α and IL-10 expressions. The pathogenic strain FL12, derived from a PRRSV infectious clone, consistently suppressed TNF-α but failed to induce IL-10 in infected swine cells. In addition, no significant IL-10 production takes place in pig tissues following infection with vFL12. Using a TNF-α promoter-reporter gene construct, we demonstrated that the viral non-structural proteins, Nsp1α and Nsp1β reduce the TNF-α promoter activity in transiently transfected cells, mainly by inhibiting the cellular transcription factors Nuclear Factor-κB (NF-κB) and Specificity Protein 1 (Sp1) respectively. Furthermore, screening of Nsp1α mutant constructs revealed that five amino acid residues (Gly90, Asn91, Arg97, Arg100, and Arg124) are important for inhibiting the TNF-α promoter activity. Nsp1α also reduced TNF-α protein levels in an in vitro translation assay, and Gly90 was important for this activity. Screening of Nsp1β mutant constructs showed that multiple amino acid stretches in all domains are important for inhibiting the TNF-α promoter activity. We obtained two mutant vFL12 strains with substitutions at Nsp1αGly90 and Nsp1β70SMVRE74 positions. Both mutant viruses increased TNF-α mRNA levels at early times after infection when compared to parental vFL12 strain in infected macrophages. However, only Nsp1α mutant virus induced higher TNF-α protein levels when compared to vFL12 in infected macrophage cultures. Moreover, Nsp1β mutant virus was attenuated in infected pigs. In summary, we have identified proteins that regulate the expression of TNF-α. These results may be useful for designing novel prophylactics and therapeutics for PRRSV.

Adviser: Fernando A. Osorio

]]> Sakthivel Subramaniam CHARACTERIZATION OF THE SERO-REACTIVITY OF PROTEINS MAP1152 AND MAP1156 FROM <i>MYCOBACTERIUM AVIUM</i> SUBSPECIES <i>PARATUBERCULOSIS</i> http://digitalcommons.unl.edu/vetscidiss/6 http://digitalcommons.unl.edu/vetscidiss/6 Tue, 30 Nov 2010 08:55:09 PST Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease (JD) in ruminants. Development of genetic tools and completion of the MAP genome sequencing project expanded opportunities for antigen discovery. In this thesis, I review the current trends in diagnosis and disease control of JD and present the results of the studies on the seroreactivity of two proteins encoded for by the MAP1152-MAP1156 gene cluster. MAP1152 encodes for a PPE protein and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Maltose-binding protein (MBP) tagged recombinant MAP proteins were purified from Escherichia coli. Western immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of immunized mice and rabbits, and naturally infected cattle. MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme linked immunosorbent assay (ELISA) for the recombinant proteins was developed and used to test pre-classified positive and negative serum samples from naturally infected and non-infected cattle. Samples, with one exception, displayed no seroreactivity against MBP-LacZ (P > 0.05), the negative control antigen. MAP1152 displayed seroreactivity against all positive sera, but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between non- infected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, MAP infected cattle mount a humoral response to both MAP1152 and MAP1156. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immuno-pathogenesis of JD.

Adviser: Raul G. Barletta

]]> Avery L. Paulson <i>Staphylococcus aureus</i> Virulence Factors Synthesis is Controlled by Central Metabolism http://digitalcommons.unl.edu/vetscidiss/5 http://digitalcommons.unl.edu/vetscidiss/5 Fri, 22 Oct 2010 10:42:00 PDT Staphylococcus aureus is a versatile pathogen that can survive in diverse host environments. This versatility depends on its ability to sense nutrients and respond by modulating gene expression, including the synthesis of virulence determinants. In addition to its ability to synthesize virulence factors, the capacity of S. aureus to form biofilms is an important mediator of virulence in certain infections. Biofilms are a complex aggregation of bacteria commonly encapsulated by an adhesive exopolysaccharide matrix (polysaccharide intercellular adhesin; PIA). To study S. aureus biofilm formation, we assessed the metabolic requirements of S. aureus growing in a biofilm and found the bacteria extracted glucose and accumulated lactate, acetate, formate, and acetoin. Additionally, S. aureus selectively extracted six amino acids from the culture medium (serine, proline, arginine, glutamine, glycine, and threonine). The major staphylococcal exopolysaccharide, PIA, is synthesized when the tricarboxylic acid (TCA) cycle is repressed. To better understand TCA cycle-dependent regulation of PIA and virulence factor synthesis in S. aureus, we artificially induced the TCA cycle by limiting its ability to exogenously acquire a TCA cycle-derived amino acid (i.e., glutamine) by inactivating the glutamine permease gene (glnP) and assessed the effects on biofilm formation and virulence factor synthesis. We found that inactivation of this major glutamine transporter increased TCA cycle activity, transiently decreased PIA synthesis, and significantly reduced in vivo virulence in a rabbit endocarditis model, establishing a causal relationship between TCA cycle activity and virulence factor synthesis. This causal relationship between the TCA cycle and virulence factor synthesis suggests there are regulatory proteins connecting metabolism and the regulation of virulence factor synthesis. This regulation is likely to occur when a metabolite-responsive regulator responds to changes in TCA cycle associated biosynthetic intermediates, the redox status, and/or ATP. In related work, NMR metabolomic analysis of S. epidermidis indicated that TCA cycle stress altered the intracellular concentration of ribose. Using this information, three putative ribose-responsive RpiR-family regulators (orfs SAV0317, SAV0193 and SAV2315) were identified in S. aureus strain UAMS-1. The proteins encoded by sav0317 and sav0193 regulate hexose monophosphate shunt transcription and alter virulence factor synthesis by increasing the transcription or stability of RNAIII. These data confirm a close linkage of central metabolism and virulence factor synthesis in S. aureus and establish that this metabolic linkage can be manipulated to alter infectious outcomes.

]]> Yefei Zhu INFLUENCE OF TYPE 2 BOVINE VIRAL DIARRHEA VIRUS N<sup>PRO</sup> ON ENHANCEMENT OF BOVINE RESPIRATORY SYNCYTIAL VIRUS REPLICATION MEDIATED BY ANTAGONISM OF HOST CELL INTERFERON TYPE I RESPONSES http://digitalcommons.unl.edu/vetscidiss/4 http://digitalcommons.unl.edu/vetscidiss/4 Fri, 30 Jul 2010 10:18:03 PDT Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, Family Flaviviridae. The virus can infect many species of animals of the order Artiodactyla. The BVDV genome encodes an auto protease, Npro, that degrades interferon regulatory factor-3 (IRF-3) reducing type I interferon (IFN-I) production from host cells. Bovine respiratory syncytial virus (BRSV) is a member of the genus Pneumovirus, Family Paramyxoviridae. Concurrent infection with BVDV and BRSV causes more severe respiratory and enteric disease than infection with either virus alone. Our hypothesis was that Npro modulates the innate immune responses to BVDV infection and enhances replication of BVDV or BRSV co-infection. The noncytopathic BVDV2 viruses NY93/c N- Npro 18 EGFP (a mutant with modified Npro fused with enhanced green fluorescent protein), NY93 infectious clone (NY93/c), wild-type NY93-BVDV2 (NY93-wt), and BRSV were evaluated in this study. The objectives of this study were: (1) to characterize the replication kinetics and IFN-I induction in Madin-Darby bovine kidney (MDBK) cells following infection with each of the BVDV isolates, and (2) to characterize the influence of BVDV-mediated IFN-I antagonism on enhancement of BRSV replication in bovine turbinate (BT) cells. NY93/c N- Npro 18 EGFP replicated 0.4 – 1.6 TCID50 logs lower than NY93-wt in MDBK cells. NY93/c N- Npro 18 EGFP-infected MDBK cells synthesized IFN-I significantly higher than NY93/c- and NY93-wt-infected MDBK cells. BT cells co-infected with NY93/c N- Npro 18 EGFP/BRSV or NY93-wt/BRSV were evaluated to determine the effects of co-infection on BRSV replication and IFN-I induction in BT cells. BRSV RNA levels in NY93-wt/BRSV co-infected BT cells were 2.49, 2.79, and 2.89 copy number logs significantly greater than in NY93/c N- Npro 18 EGFP/BRSV co-infected BT cells on days 5, 7, and 9 post-infection, respectively. BVDV RNA levels in NY93/c N- Npro 18 EGFP-infected BT cells were 1.64 – 4.38 copy number logs lower than in NY93-wt-infected BT cells. NY93/c N- Npro 18 EGFP single and co-infected BT cells synthesized IFN-I significantly higher than NY93-wt single and co-infected BT cells. In summary, these findings suggest: (1) NY93/c N- Npro 18 EGFP BVDV2 induced higher levels of IFN-I than BVDV2-wt and may be useful as a safer, replicating BVDV vaccine, and (2) Enhancement of BRSV infection by BVDV co-infection is mediated by antagonism of IFN-I.

]]> Abdulrahman Abdulaziz A. Alkheraif THE GLYCOPROTEINS OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS AND THEIR ROLE IN INFECTION AND IMMUNITY http://digitalcommons.unl.edu/vetscidiss/3 http://digitalcommons.unl.edu/vetscidiss/3 Wed, 28 Jul 2010 07:09:53 PDT The porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen of swine and is known to cause abortion and infertility in pregnant sows and respiratory distress in piglets. PRRSV contains a major glycoprotein (GP5) and three minor glycoproteins (GP2a, GP3, and GP4) on the virion envelope, all of which are required for infectious virus production. To study their interactions amongst each other and with a cellular receptor for PRRSV, CD163, I cloned each of the viral glycoproteins and CD163 in various expression vectors. My studies have shown that while the GP2a, GP3, and GP4 are co-translationally glycosylated, the GP5 is post-translationally glycosylated. By using co-immunoprecipitation (co-IP) assays, strong interaction was demonstrated between GP4 and GP5 proteins, although weak interactions among the other envelope glycoproteins were also detected. Further, GP4 was found to mediate interactions leading to formation of multiprotein glycoprotein complex. My results also show that GP2a and GP4 proteins are the only two GPs that specifically interact with the CD163 molecule and that glycosylation of these GPs is required for efficient interaction. Based on these studies, I have developed an interactome map of the viral GPs and CD163 and have proposed a model of the viral glycoprotein complex and its interaction with CD163. Studies reported here also show that glycan addition at residue 184 (N184) of GP2a, and residues N42, N50, and N131 of GP3 is essential for recovery of infectious virus. Although single site glycosylation mutants of GP4 had no effect on infectious virus production, introduction of double mutations was lethal. The loss of glycan moieties of GP2a, GP3, and GP4 proteins had no effect on host neutralizing antibody production. Overall, I conclude that the PRRSV glycoproteins are co-translationally and post-translationally glycosylated, the GP4 protein is central to mediating interglycoprotein interactions, and along with GP2a, serves as the viral attachment protein that is responsible for interactions with the viral receptor, CD163. Further, glycosylation of GP2a, GP3, and GP4 proteins is required for infectious virus production, efficient interaction with CD163, but does not play any role in neutralizing antibody response in infected animals.

]]> Phani B. Das Tactics for Identifying and Eliminating Tritrichomonas foetus from Infected Beef Herds http://digitalcommons.unl.edu/vetscidiss/2 http://digitalcommons.unl.edu/vetscidiss/2 Wed, 21 Apr 2010 07:47:54 PDT The protozoan, Tritrichomonas foetus (TF), has been recognized as a cause of bovine infertility for more than 100 years (Skirrow and BonDurant, 1988). As an obligate parasite of the bovine reproductive tract its control and eradication seems achievable (Harding, 1950). However, this disease continues to trouble US cattle producers and a recent epidemic in the Western US has lead to increased interest in research and regulatory efforts (Cima, 2009).

Outbreak investigations were carried out on three Nebraska ranches to assess the efficiency of currently available diagnostic tests, culture, gel polymerase chain reaction (PCR), and real time PCR (rtPCR), in identifying TF infected bulls in known TF infected herds with the following objectives:

(1) to compare the agreement of the three assays for classifying the status of individual preputial specimens.

(2) to compare the agreement of the three assays in identifying TF infected bulls based on three sequential samples.

(3) to correlate cow herd pregnancy percentages with TF herd bull prevalence.

Comparisons of diagnostic tests were conducted using Cohen’s Kappa statistic and McNemar’s paired sample Chi square test p values. Simple linear regression was used to assess the relationship between non-pregnancy percentages and prevalence of TF positive bulls.

No significant differences between culture and gel PCR for individual specimen and bull TF classification were found. Real time PCR had a high rate of apparent false positives relative to culture and gel PCR for individual specimen and bull TF classification. However, all assays required multiple, sequential specimens to adequately identify all TF infected bulls in the study herds. Cow non-pregnancy rates correlated linearly with TF positive bull prevalence.

These studies indicate similar diagnostic assay performance for culture, gel PCR, and real time PCR which suggests opportunities for improved TF control may be found by focusing on pre-analytical aspects of diagnostic TF detection such as consistent bull identification, optimization of specimen collection techniques, and pre-incubation specimen handling factors.

]]> Jeff D. Ondrak