Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.

<p><i>In vitro</i> production of embryos allows efficient management of herd genetics, reduction of disease impact, and if used in combination with other reproductive technologies it could aid in preserving the threatened genetic diversity of swine. <i>In vitro</i> maturation (IVM) is identified as a deficient step in porcine in vitro production (IVP) of embryo systems, which decreases the overall success of IVP. There are problems encountered in each step of IVP; chromosomal abnormalities and decreased cell numbers in blastocysts during <i>in vitro</i> culturing (IVC), and low monospermic fertilization rates during <i>in vitro</i> fertilization (IVF) may be a result of insufficient IVM. As an addition to maturation media, porcine follicular fluid (pFF) can affect IVM. Estrogen can be found in high concentrations in pFF; possibly contributing to the effects seen when pFF is added to IVM. The objective of this thesis was to investigate the effects of estrogen supplementation during IVM on IVP of porcine embryos.</p> <p>The first objective was to evaluate the <i>in vitro</i> maturation rates of porcine oocytes in two maturation media: protein-free and 10% pFF supplemented. Nuclear maturation of oocytes was evaluated using Lamin/Dapi staining of oocytes matured in protein-free and 10% pFF maturation media to ensure the efficiency of the protein-free media. Protein-free and 10% pFF media mature oocytes at similar rates (91% and 89% respectively).</p> <p>The transcripts within the oocyte can be altered based on the <i>in vitro</i> maturation environment, so the second objective was to observe the expression of four chosen maternal effect genes: Basonuclin-1 (<i>BNC1</i>), Nucleoplasmin 2 (<i>NPM2</i>), Zygote arrest 1 (<i>ZAR1</i>), and Tripartite-motif protein-24 (<i>TRIM24</i>), using oocytes matured in 50 ng/ml, 100 ng/ml, or 1000 ng/ml of estradiol 17-&beta; (E₂), 10% pFF, or protein-free maturation media. Expression of maternal effect genes, was shown by the &Delta;Ct (cycle threshold) values, obtained from the difference between the Ct values of the normalizing gene (<i>GAPDH</i>) and the genes of interest evaluated through QRT-PCR. Values of &Delta;Ct were analyzed in place of fold change to avoid data manipulation. The &Delta;Ct expression of <i>TRIM24</i> in 0 ng/ml E₂ maturation medium and the 10% pFF maturation medium were significantly different (p<0.05) from the non-matured control, the other maternal determinant genes did not differ in their expression under any treatment.</p> <p>We hypothesized that estradiol's effects on IVM would be evident when analyzing cleavage and blastocyst formation rates. Cleavage and blastocyst formation rates were examined following <i>in vitro</i> fertilization of oocytes matured in 100 ng/ml E₂, 10% pFF, or a protein-free maturation medium to investigate the effect of estradiol on IVP embryos. Cleavage rates for the E₂ (n= 252; 60.2%) or 10% pFF (n= 256; 55.7%) additions to the maturation media did not differ (p>0.05) when compared to the protein-free maturation media (n=264; 54.9%). Both 10% pFF and E<syb>2</sub> groups had significantly higher blastocyst formation rates (p&le;0.05) than the protein-free maturation media (n=264; 3.5%), although no statistical difference was observed between the blastocyst formation rates of the 10% pFF (n=256; 12.4%) and E2 (n=252; 14.6%) groups.</o> <p>As a final study, the global gene expression of oocytes matured in a control protein-free media and the protein-free media supplemented with 100 ng/ml E₂ or 10% pFF was investigated using microarray analysis. Genes were not differentially expressed among the matured groups with the outlined threshold values of -2 &ge; log2(fold change) &ge; 2, and adjusted p-value &le;0.05. A total of 16 differentially expressed genes between the non-matured and all matured groups exceeded this threshold. Of these genes, 6 are novel transcribed regions with evidence of being an embryonic EST, and 1 is a novel protein-coding gene. The other genes are FBJ murine osteosarcoma viral oncogene homolog (<i>FOS</i>), Vimentin (<i>VIM</i>), Capthesin C (<i>CTSC</i>), Selenium binding protein 1 (<i>SELENBP1</i>), Poly(A) binding protein cytoplasmic 1 (<i>PABPC1</i>), Tissue factor pathway inhibitor 2 (<i>TFPI2</i>), Cysteine-rich, angiogenic inducer 61 (<i>CYR61</i>), Acyl-CoA synthetase long-chain family member 6 (<i>ACSL6</i>), and Phospholipase A2 group VII (<i>PLA2G7</i>).</p> <p>In conclusion, successful nuclear maturation of oocytes derived of prepubertal gilt abbatoire derived ovaries can be achieved without pFF or hormone supplementation. The expression of maternal determinant genes is not affected in a dose dependant manner, and removal of E₂ or supplementation of pFF during maturation may alter the expression of <i>TRIM24</i> from the non-matured control; where no other maternal effect gene changes through maturation. Estradiol has a similar effect as pFF during <i>in vitro</i> maturation of porcine oocytes as seen by cleavage and blastocyst formation rates. And media does not affect the global gene expression of porcine oocytes, though there is a temporal control of gene expression through maturation.</p>

F4 positive enterotoxigenic Escherichia coli (ETEC) is the most common pathogen causing neonatal diarrhea in piglets. The pathogenesis requires the attachment of ETEC to the intestinal brush border, mediated by F4 fimbria. Colostral anti-F4 antibodies and some non-immunoglobulin porcine skim milk proteins can bind F4 and prevent colonization and infection by F4-positive ETEC. Little is known, however, about the F4-binding ability of porcine milk fat globule membrane (MFGM) proteins. In addition, the knowledge of the absorption of porcine colostral proteins into the blood of neonatal piglets is limited, despite the well accepted concept that in neonatal piglets, protein absorption from the intestine is non-selective.<p> In this study, the ability of porcine MFGM proteins to bind purified F4ac (one of the three subtypes of F4 fimbriae) was investigated. Porcine MFGM proteins were first separated by 2D SDS-PAGE and subsequently identified by mass spectrometry. Overlay western Blot was then employed to demonstrate the interaction between porcine MFGM proteins and purified F4ac. Several proteins from porcine MFGM reacted with F4ac, and of these, lactadherin, butyrophilin, adipophilin, and acyl-CoA synthetase 3 reacted strongly. The biological function of these proteins in vivo was not investigated but it is possible that their interaction with F4ac positive ETEC interferes with bacterial attachment and colonization. In order to investigate protein absorption by neonatal piglets after natural suckling, the protein profiles of the plasma of pre-suckling and 24 h post-suckling neonatal piglets were studied by 2D SDS-PAGE. Those plasma proteins that increased prominently after suckling were then identified by mass spectrometry. Only immunoglobulins were unequivocally determined to be absorbed, because they were absent before suckling and present in large quantity in plasma 24 h after suckling. The absorption of other colostral proteins was either equivocal or not detectable by our detection methods. These results suggest that, unlike immunoglobulins, major non-immunoglobulin proteins in porcine colostrum may not be absorbed into systemic circulation in substantial amounts.

Dendritic cells (DCs) are potent antigen presenting cells (APCs) that link the innate and adaptive immune system by their unique ability to induce and direct immune responses towards various T helper (Th)-type of immune responses such as Th1-, Th2-, Th9-, Th17-, Th22- or T regulatory (TR). The type of Th response generated very much depends on the nature of the antigen encountered and allows for an effective and proficient immune response. For example, Th1 responses are used to clear intracellular pathogens while Th2 responses are needed to clear extracellular pathogens The ability to specifically modulate Th-responses is an area of intense research, as it allows for the development of more effective vaccines and immunotherapeutics. Immunomodulation of DCs is one strategy by which specific Th-type immune responses may be tailored. Current research is focused on identifying agents that have the capacity to immunomodulate DCs such as host defense peptides (HDPs). Apart from their anti-microbial activities, HDPs have a number of immune functions including recruitment and subsequent activation of DCs. The goal of this study was to examine the immunomodulatory effects of HDPs on porcine DC functions. This research was part of a larger multinational research project to develop a novel adjuvant platform for single-immunization vaccines against pertussis in neonates. The pig model was used for this research because of its physiological similarities to humans and the recently developed pertussis infection model in young piglets. A series of experiments was conducted to characterize and describe porcine DC functions. Two subsets of DCs were successfully characterized and tested for their response to stimulation with HDPs. Initial results demonstrated a minimal effect of HDPs on DC functions, therefore we expanded the number of HDPs used to include both synthetic derivatives of HDPs known as innate defense regulators (IDRs) and naturally- occurring HDPs. We examined these effects on peripheral blood mononuclear cells (PBMC) in vitro and found that HDPs induce expression of the chemokine interleukin (IL)-8, which resulted in PBMC recruitment in vitro. We then proceeded to evaluate the HDPs in vivo by intradermally administering them into the flank of pigs. Surprisingly, treatment with the HDPs did not result in recruitment of neutrophils in vivo. We also examined the effects of formulating IDR-1002 as an adjuvant with the academic antigen Keyhole Limpet Hemocyanin (KLH) on the development of KLH-specific immune responses in vaccinated pigs. While there was no difference in antibody titers between vaccinated and control animals, we found that co-formulation with IDR-1002 decreased both antigen-specific and mitogen-induced proliferation in KLH/IDR-1002 vaccinated animals as long as four weeks post-treatment. These results demonstrate that specific IDRs can suppress certain aspects of the pro-inflammatory immune response making them potentially highly versatile tools to modulate and tailor the immune response in disease states characterized by a pro-inflammatory component.

La polispermia (entrada de más de un espermatozoide al ovocito) es una condición patológica en mamíferos ya que impide el desarrollo embrionario. En la especie porcina, la polispermia es un problema común y aún sin resolver en los sistemas de fecundación in vitro (FIV). Los gránulos corticales (GCs) de los ovocitos de mamíferos estan implicados en el bloqueo de la polispermia, sin embargo, poco se sabe acerca de la composición y función de estas organelas. Esta ampliamente descrito que las moléculas liberadas de los GCs durante la fecundación o activación ovocitaria mediante estimulación química o eléctrica producen importantes modificaciones, particularmente en la zona pelúcida (ZP), el espacio perivitelino y, probablemente, el oolema del ovocito. Estas modificaciones repercuten directamente en el bloqueo de la polispermia. El conocimiento del contenido de los GCs y proteínas del oolema así como su papel durante la fecundación en la especie porcina es escaso. La mayoría de la información que se tiene en la actualidad se ha obtenido del modelo murino. En la presente tesis doctoral, investigamos la presencia de posibles proteínas de los GCs en ovocitos porcinos así como la presencia de metaloproteasas ADAM-10 y ADAM-17 en ovocitos porcinos y bovinos. Para ello, este estudio se dividió en tres apartados: / Polyspermy (entering of more than a spermatozoon into the oocyte) is a pathological condition in mammals since it avoids the normal embryonic development. In the pig species, Polyspermy is a common problem still unsolved in the current systems of in vitro fertilization (IVF). The cortical granules (CGs) from mammal's oocytes are involved in the block to polyspermy. However, little is known about the composition and function of these organelles. It is widely described that the molecules released of the CGs during the fertilization or oocyte activation, by means of chemical or electrical stimulation, produce important modifications, particularly in the zona pelucida (ZP), the periviteline space and, probably, oolema of the oocyte. These modifications have a direct role in the block to polyspermy. The knowledge about the content of the CGs and the oolema proteins of as well as their role during fertilization in pigs is still scarce. The majority of the information that we currently have it has been obtained from the murine model. In the present Doctoral Thesis, we investigated the presence of possible proteins of the CGs in pig oocytes as well as the presence of metaloproteases ADAM-10 and ADAM-17 in pig and bovine oocytes. For that, this study was divided into three sections

Respiratory diseases are a major cause of human morbidity and mortality and are a leading cause of economic loss to livestock producers. The respiratory tract is constantly in contact with dust, bacteria, fungi, viruses and other pathogenic agents that are found in the air. Normally, the body has the ability to clear these foreign particles. However, physiological and environmental stresses can impair airway defense mechanisms resulting in establishment of pulmonary infections. The microbes and their products engage various receptors in the lung to activate epithelium, endothelium, macrophages, neutrophils and other cells. The activation of inflammatory cascade in the lung results in recruitment of neutrophils, damage to air-blood barrier and development of edema. Although there have been significant advances in our understanding of mechanisms of lung inflammation, there have been a lack of any significant advances in the development of new therapeutics to manage lung disease, which may suggest that our understanding of the inflammatory mechanisms is still incomplete. Pentraxin 3 (PTX3) is an innate immune protein which has been implicated in a diverse range of inflammatory processes, such as recruitment of cells and production of cytokines. PTX3 is an acute phase protein, with low or undetectable levels in the circulation of healthy humans and animals, and rapid, dramatic increase in inflammatory diseases. The expression and function of this protein has not been characterized in the lungs of domestic animal species. Because of potential implications of PTX3 in lung inflammation, I studied the expression of PTX3 in normal and inflamed lungs of calves, pigs, horses, foals and humans. Lungs from all of these species showed expression of PTX3 in airway epithelium, alveolar septa, vascular endothelium and inflammatory cells. Western blot performed on homogenates from normal and inflamed lungs from calves and pigs show an increased expression of PTX3 in inflamed lungs (P<0.05). Because protein function is influenced by its location in the cell, I clarified the subcellular expression of PTX3 with immuno-electron microscopy on normal and inflamed calf and horse lungs. PTX3 was localized on pulmonary intravascular macrophages, monocytes, neutrophils and, unexpectedly, platelets. PTX3 was also present in the nuclei of neutrophils, monocytes and pulmonary intravascular macrophages. Neutrophils are critical regulators of acute lung inflammation. Having observed PTX3 in neutrophils, I investigated the effect of E. coli lipopolysaccharide-induced activation on PTX3 in neutrophils in vitro. Neutrophils challenged with E. coli LPS were examined at 30, 60, 90 and 120 minutes after the treatment. Normal peripheral blood neutrophils showed PTX3 expression. Neutrophils activated with LPS appeared ruffled and showed loss of PTX3 expression at 30 minutes followed by recovery of the expression. Western blots performed on normal and activated neutrophil homogenates did not show any differences (P=0.05). Collectively, the data show PTX3 in normal and inflamed lungs across multiple species. PTX3 was also detected in normal and activated neutrophils. While the function of intriguing localization of PTX3 in the nuclei as well as in platelets is not known, the similarity of expression across the species suggest a role for PTX3 in lung inflammation.

<p><i>In vitro</i> production of embryos allows efficient management of herd genetics, reduction of disease impact, and if used in combination with other reproductive technologies it could aid in preserving the threatened genetic diversity of swine. <i>In vitro</i> maturation (IVM) is identified as a deficient step in porcine in vitro production (IVP) of embryo systems, which decreases the overall success of IVP. There are problems encountered in each step of IVP; chromosomal abnormalities and decreased cell numbers in blastocysts during <i>in vitro</i> culturing (IVC), and low monospermic fertilization rates during <i>in vitro</i> fertilization (IVF) may be a result of insufficient IVM. As an addition to maturation media, porcine follicular fluid (pFF) can affect IVM. Estrogen can be found in high concentrations in pFF; possibly contributing to the effects seen when pFF is added to IVM. The objective of this thesis was to investigate the effects of estrogen supplementation during IVM on IVP of porcine embryos.</p> <p>The first objective was to evaluate the <i>in vitro</i> maturation rates of porcine oocytes in two maturation media: protein-free and 10% pFF supplemented. Nuclear maturation of oocytes was evaluated using Lamin/Dapi staining of oocytes matured in protein-free and 10% pFF maturation media to ensure the efficiency of the protein-free media. Protein-free and 10% pFF media mature oocytes at similar rates (91% and 89% respectively).</p> <p>The transcripts within the oocyte can be altered based on the <i>in vitro</i> maturation environment, so the second objective was to observe the expression of four chosen maternal effect genes: Basonuclin-1 (<i>BNC1</i>), Nucleoplasmin 2 (<i>NPM2</i>), Zygote arrest 1 (<i>ZAR1</i>), and Tripartite-motif protein-24 (<i>TRIM24</i>), using oocytes matured in 50 ng/ml, 100 ng/ml, or 1000 ng/ml of estradiol 17-&beta; (E₂), 10% pFF, or protein-free maturation media. Expression of maternal effect genes, was shown by the &Delta;Ct (cycle threshold) values, obtained from the difference between the Ct values of the normalizing gene (<i>GAPDH</i>) and the genes of interest evaluated through QRT-PCR. Values of &Delta;Ct were analyzed in place of fold change to avoid data manipulation. The &Delta;Ct expression of <i>TRIM24</i> in 0 ng/ml E₂ maturation medium and the 10% pFF maturation medium were significantly different (p<0.05) from the non-matured control, the other maternal determinant genes did not differ in their expression under any treatment.</p> <p>We hypothesized that estradiol's effects on IVM would be evident when analyzing cleavage and blastocyst formation rates. Cleavage and blastocyst formation rates were examined following <i>in vitro</i> fertilization of oocytes matured in 100 ng/ml E₂, 10% pFF, or a protein-free maturation medium to investigate the effect of estradiol on IVP embryos. Cleavage rates for the E₂ (n= 252; 60.2%) or 10% pFF (n= 256; 55.7%) additions to the maturation media did not differ (p>0.05) when compared to the protein-free maturation media (n=264; 54.9%). Both 10% pFF and E<syb>2</sub> groups had significantly higher blastocyst formation rates (p&le;0.05) than the protein-free maturation media (n=264; 3.5%), although no statistical difference was observed between the blastocyst formation rates of the 10% pFF (n=256; 12.4%) and E2 (n=252; 14.6%) groups.</o> <p>As a final study, the global gene expression of oocytes matured in a control protein-free media and the protein-free media supplemented with 100 ng/ml E₂ or 10% pFF was investigated using microarray analysis. Genes were not differentially expressed among the matured groups with the outlined threshold values of -2 &ge; log2(fold change) &ge; 2, and adjusted p-value &le;0.05. A total of 16 differentially expressed genes between the non-matured and all matured groups exceeded this threshold. Of these genes, 6 are novel transcribed regions with evidence of being an embryonic EST, and 1 is a novel protein-coding gene. The other genes are FBJ murine osteosarcoma viral oncogene homolog (<i>FOS</i>), Vimentin (<i>VIM</i>), Capthesin C (<i>CTSC</i>), Selenium binding protein 1 (<i>SELENBP1</i>), Poly(A) binding protein cytoplasmic 1 (<i>PABPC1</i>), Tissue factor pathway inhibitor 2 (<i>TFPI2</i>), Cysteine-rich, angiogenic inducer 61 (<i>CYR61</i>), Acyl-CoA synthetase long-chain family member 6 (<i>ACSL6</i>), and Phospholipase A2 group VII (<i>PLA2G7</i>).</p> <p>In conclusion, successful nuclear maturation of oocytes derived of prepubertal gilt abbatoire derived ovaries can be achieved without pFF or hormone supplementation. The expression of maternal determinant genes is not affected in a dose dependant manner, and removal of E₂ or supplementation of pFF during maturation may alter the expression of <i>TRIM24</i> from the non-matured control; where no other maternal effect gene changes through maturation. Estradiol has a similar effect as pFF during <i>in vitro</i> maturation of porcine oocytes as seen by cleavage and blastocyst formation rates. And media does not affect the global gene expression of porcine oocytes, though there is a temporal control of gene expression through maturation.</p>

F4 positive enterotoxigenic Escherichia coli (ETEC) is the most common pathogen causing neonatal diarrhea in piglets. The pathogenesis requires the attachment of ETEC to the intestinal brush border, mediated by F4 fimbria. Colostral anti-F4 antibodies and some non-immunoglobulin porcine skim milk proteins can bind F4 and prevent colonization and infection by F4-positive ETEC. Little is known, however, about the F4-binding ability of porcine milk fat globule membrane (MFGM) proteins. In addition, the knowledge of the absorption of porcine colostral proteins into the blood of neonatal piglets is limited, despite the well accepted concept that in neonatal piglets, protein absorption from the intestine is non-selective.<p> In this study, the ability of porcine MFGM proteins to bind purified F4ac (one of the three subtypes of F4 fimbriae) was investigated. Porcine MFGM proteins were first separated by 2D SDS-PAGE and subsequently identified by mass spectrometry. Overlay western Blot was then employed to demonstrate the interaction between porcine MFGM proteins and purified F4ac. Several proteins from porcine MFGM reacted with F4ac, and of these, lactadherin, butyrophilin, adipophilin, and acyl-CoA synthetase 3 reacted strongly. The biological function of these proteins in vivo was not investigated but it is possible that their interaction with F4ac positive ETEC interferes with bacterial attachment and colonization. In order to investigate protein absorption by neonatal piglets after natural suckling, the protein profiles of the plasma of pre-suckling and 24 h post-suckling neonatal piglets were studied by 2D SDS-PAGE. Those plasma proteins that increased prominently after suckling were then identified by mass spectrometry. Only immunoglobulins were unequivocally determined to be absorbed, because they were absent before suckling and present in large quantity in plasma 24 h after suckling. The absorption of other colostral proteins was either equivocal or not detectable by our detection methods. These results suggest that, unlike immunoglobulins, major non-immunoglobulin proteins in porcine colostrum may not be absorbed into systemic circulation in substantial amounts.

Porcine <i>Circovirus</i> 2 (PCV2) associated diseases (PCVAD) cause economic loss to the global swine industry. Control measures for PCVAD largely depend on the use of PCV2 vaccines. The available commercial PCV2 vaccines contain either inactivated whole virus particles or recombinant PCV2 capsid protein. These preparations most likely contain varying amounts of immune-irrelevant proteins that can cause adverse injection site reactions, with compromised efficacy due to alteration of protective immune epitopes arising during the viral inactivation process. Other constraints include high production cost attributed to propagation of slow growing virus and expression and extraction of recombinant proteins, a requirement for adjuvants, and the induction of a Th2-biased immune response. Hence, development of new PCV2 vaccines is necessary.<p> There are two recommended PCV2 vaccination strategies. They are i. vaccinating sows, which relies on the passive transfer of maternal immunity to offspring, and ii. immunizing young piglets to induce an active immune response. The piglet vaccination has been shown to confer better protection from mortality. Maternal antibody interference to the induction of an active immune response is an obstacle when piglets are vaccinated at an early age. Can we sidestep this maternal antibody interference? To address this issue, I investigated whether a prototypical PCV2 vaccine, parenterally administered, could override maternally-derived PCV2 antibodies in seropositive piglets. The results of this study were not conclusive. However, they laid the foundation for future studies based upon using varying levels of vaccine antigen with different adjuvants, and administered to piglets with defined maternally derived PCV2 antibodies.<p> Subsequently, I examined if a new PCV2 vaccine candidate comprised of bacteriophage lambda particles displaying part of the PCV2 capsid protein could induce anti-PCV2 immunity. Initial experiments showed that pigs do not have pre-existing anti-lambda antibodies and thus will not neutralize display particles used as a vaccine at primary vaccination. I produced and characterized lambda phage particles displaying four immunodominant regions of porcine circovirus 2 (PCV2) capsid protein fused to the lambda capsid protein D i.e., D-CAP, phage display particles. Expression of D-CAP in <i>Escherichia coli</i> (<i>E. coli</i>) and its presence in the vaccine preparation was shown by ELISA and Western blots using anti-PCV2 polyclonal antiserum from a gnotobiotic pig. The vaccine, lambda particles displaying PCV2 capsid protein immunogenic epitopes fused to lambda D protein (LDP-D-CAP), administered without an adjuvant induced both humoral and cellular immunity to PCV2 in conventional pigs, as shown by ELISA, Western blots, virus neutralization assay and delayed type hypersensitivity (DTH) reactions. This work produced the first potential phage vaccine to PCV2. In order to further investigate the feasibility of using the lambda display technology. I produced and characterized two additional lambda display particle preparations, LDP-D-FLAG and LDP-D-GFP, displaying a FLAG tag and the green fluorescent proteins, respectively.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea to pigs at all ages, resulting in high mortality rate of 80-100% in piglets less than one week old. Within one year after the outbreak in April 2013, PEDV has rapidly spread in the US and causes the loss of over 10% of the US pig population. Monoclonal antibody (mAb) is a key reagent for rapid diagnosis of PEDV infection. In this study, we produced a panel of mAbs against nonstructural protein 8 (nsp8), spike(S) protein, and nucleocapsid (N) protein of PEDV. Four mAbs were selected, which can be used in various diagnostic assays, including indirect immunofluorescence assay (IFA), enzyme-linked immunoabsorbent assay (ELISA), Western Blot, immunoprecipitation (IP), immunohistochemistry (IHC) test and fluorescence in situ hybridization (FISH). The mAb 51-79 recognizes amino acid (aa) 33-60 of nsp8, mAb 70-100 recognizes aa1371-1377 of S2 protein, and mAb 66-155 recognizes aa 241-360 of N protein, while mAb 13-519 is conformational. Using the mAb70-100, the immunoprecipitated S2 fragment was examined by protein N-terminal sequencing, and cleavage sites between S1 and S2 was identified. In addition, this panel of mAbs was further applied to determine the infection site of PEDV in the pig intestine. IHC test result showed that PEDV mainly located at the mid jejunum, distal jejunum and ileum. Results from this study demonstrated that this panel of mAbs provides a useful tool for PEDV diagnostics and pathogenesis studies.