Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus associated disease (PCVAD), an economically important swine disease that causes wasting in pigs 5-18 weeks of age. There exist two different types of porcine circoviruses: porcine circovirus type 1 (PCV1) was discovered as a contaminant of porcine kidney (PK-15) cells and was determined to be nonpathogenic in swine; whereas porcine circovirus type 2 (PCV2) is pathogenic. A recently released vaccine for PCVAD was generated by inserting the gene encoding the immunogenic capsid protein of PCV2 into the genetic backbone of the non-pathogenic PCV1. This chimeric PCV vaccine, called PCV1-2, was shown to induce protective immunity against PCV2 infection in pigs. The vaccine is currently on the market in a killed form. In order to develop a live version of the vaccine, the genetic stability of the chimeric PCV1-2 vaccine virus was investigated by in vitro and in vivo passaging of the vaccine virus. In vitro passaging of the PCV1-2 vaccine virus was done in a porcine kidney PK-15 cell line. Cells were infected with the PCV1-2 vaccine virus and then serially passaged 11 times. The passaged vaccine viruses recovered from passages 5 and 11 were sequenced, and the sequences were compared to that of the original PCV1-2 vaccine virus. The in vitro serial passage result showed that no mutation occurred during the 11 in vitro passages. The in vivo passaging was done using specific-pathogen-free (SPF) pigs. In in vivo "passage 1", nine piglets were divided into 3 groups of 3 each: group 1 each inoculated with 200ug of PCV1-2 plasmid, group 2 each with 1Ã 103 TCID50 live PCV1-2 vaccine virus, and group 3 each with 3ml phosphate buffered saline (PBS) buffer as a control. One pig from each group was necropsied at 14, 21, and 28 days post-inoculation (DPI), respectively. A panel of tissue samples including lymph nodes and thymus were collected from each pig. Tissue homogenates from DPI 28 that were positive by PCR for PCV1-2 DNA were used to inoculate new piglets in the in vivo passage 2 experiment. Viruses recovered from passage 2 pigs were subsequently used for inoculation in the in vivo passage 3 experiment. The PCV1-2 vaccine virus DNA from pigs in each passage was amplified and sequenced. The results of the in vivo serial passage experiment showed that, after 3 passages of the PCV1-2 vaccine virus in pigs, there were no new mutations in the viruses recovered from pigs. The PCV1-2 vaccine contained an introduced marker mutation at amino acid position number 79, which is in the capsid region. During the in vivo passaging of the vaccine virus in pigs, this marker mutation quickly reverted back to its original nucleotide. This marker back mutation occurred between DPI 21 and DPI 28 of passage 1 in the PCV1-2 live vaccine virus group, and between DPI 28 of passage 1 and DPI 14 of passage 2 in the PCV1-2 vaccine plasmid group, and remained stable throughout the reminder of the in vivo study. Based upon the results from this study, we conclude that the PCV1-2 chimeric vaccine virus is genetically stable in vitro and in pigs, and thus should serve as a good candidate for a live vaccine against PCV2. / Master of Science

Research has demonstrated that porcine circovirus type 2 (PCV2) can be shed into boar semen, raising the possibility that artificial insemination may be an important route by which disease associated with PCV2 is transmitted. The objective of this experiment was to determine the effect of vaccination against PCV2 on ejaculate characteristics and PCV2-specific antibody titers in serum of PCV2-positive boars viremia and viral shedding in semen. Semen and blood samples were collected weekly from week 0 to week 8. After collections at week 0, boars were vaccinated with a commercial vaccine against PCV2 (n = 5) (Suvaxyn PCV2 One dose; Fort Dodge Animal Health, Fort Dodge, IA) or served as controls and received 2 ml 0.9% saline (n = 5). Sperm concentrations and characteristics of sperm motility were assessed using a computer-assisted sperm analysis system (Hamilton Thorne Research, Beverly, MA) and sperm morphology was evaluated after staining using light microscopy. The PCV2 antibody titers were determined in serum using an ELISA (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). The genomic copy number of PCV2 DNA in serum and semen was determined by PCR (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). There were no effects of treatment or treatment by week on semen characteristics (P > 0.05). An effect of treatment by week was detected for serum antibody titers (P < 0.01). Compared with controls, antibody titers in vaccinated boars tended to be greater at week 0 (1.13 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P = 0.09) and were greater at week 2 (1.15 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P < 0.05) but lesser at week 7 (1.01 ± 0.05 titer/ml vs 1.23 ± 0.05 titer/ml; P < 0.01) and tended to be lesser at week 8 (1.05 ± 0.05 titer/ml vs 1.17 ± 0.05 titer/ml; P = 0.07). There were no effects of treatment, week, or treatment by week for serum genomic copy number of PCV2 DNA (P > 0.1). An effect of week was detected for semen genomic copy number of PCV2 DNA (P < 0.04). During week 3, PCV2 genomic copy number was at its greatest numerical value, however, semen PCV2 genomic copy number was at its lowest point. This was followed by an increase in semen PCV2 genomic copy number during week 7. This increase could be related to the increase in viral shedding in the serum. In summary, vaccination against PCV2 can lower antibody titers when given post-infection and has no effect on indicators of semen fertility. Vaccination also can decrease the length of reoccurring infection by decreasing the length of viral shedding in serum. / Master of Science

Yes / Peripheral nerve injuries affect a large proportion of the global population, often causing significant morbidity and loss of function. Current treatment strategies include the use of implantable nerve guide conduits (NGC's) to direct regenerating axons between the proximal and distal ends of the nerve gap. However, NGC's are limited in their effectiveness at promoting regeneration Current NGCs are not suitable as substrates for supporting either neuronal or Schwann cell growth, as they lack an architecture similar to that of the native extracellular matrix (ECM) of the nerve. The aim of this study was to create an acellular porcine peripheral nerve using a novel decellularisation protocol, in order to eliminate the immunogenic cellular components of the tissue, while preserving the three-dimensional histoarchitecture and ECM components. Porcine peripheral nerve (sciatic branches were decellularised using a low concentration (0.1%; w/v) sodium dodecyl sulphate in conjunction with hypotonic buffers and protease inhibitors, and then sterilised using 0.1% (v/v) peracetic acid. Quantitative and qualitative analysis revealed a ≥95% (w/w) reduction in DNA content as well as preservation of the nerve fascicles and connective tissue. Acellular nerves were shown to have retained key ECM components such as collagen, laminin and fibronectin. Slow strain rate to failure testing demonstrated the biomechanical properties of acellular nerves to be comparable to fresh controls. In conclusion, we report the production of a biocompatible, biomechanically functional acellular scaffold, which may have use in peripheral nerve repair. / Engineering and Physical Sciences Research Council. Grant Number: EPSRC EP/F500513/1

Yes / Currently available replacement heart valves all have limitations. This study aimed to produce and characterize an acellular, biocompatible porcine pulmonary root conduit for reconstruction of the right ventricular outflow tract e.g., during Ross procedure. A process for the decellularization of porcine pulmonary roots was developed incorporating trypsin treatment of the adventitial surface of the scraped pulmonary artery and sequential treatment with hypotonic Tris buffer (HTB; 10 mM Tris pH 8.0, 0.1% (w/v) EDTA, and 10 KIU aprotinin), 0.1% (w/v) sodium dodecyl sulfate in HTB, two cycles of DNase and RNase, and sterilization with 0.1% (v/v) peracetic acid. Histology confirmed an absence of cells and retention of the gross histoarchitecture. Im-munohistochemistry further confirmed cell removal and partial retention of the extracellular matrix, but a loss of collagen type IV. DNA levels were reduced by more than 96% throughout all regions of the acellular tissue and no functional genes were detected using polymerase chain reaction. Total collagen levels were retained but there was a significant loss of glycosaminoglycans following decellularization. The biomechanical, hydrody-namic, and leaflet kinematics properties were minimally affected by the process. Both immunohistochemical labeling and antibody absorption assay confirmed a lack of a-gal epitopes in the acellular porcine pulmonary roots and in vitro biocompatibility studies indicated that acellular leaflets and pulmonary arteries were not cytotoxic. Overall the acellular porcine pulmonary roots have excellent potential for development of a tissue substitute for right ventricular outflow tract reconstruction e.g., during the Ross procedure.

Transportation of swine is an emerging welfare issue, especially for piglets weaned and then immediately transported. Weaned pigs fed starter diets containing SDPP display improved growth performance. The objective of this study was to determine effects of pre-weaning SDPP on indicators of health, welfare, and performance in transported weaned pigs. Pigs were assigned to treatments: I. SDPP + transport, II. Water + transport, III. SDPP + no transport, or IV. Water + no transport. Pigs received their gavage twice daily for 5 d prior to weaning. Pigs were weaned and either transported or moved directly to the wean-to-finish barn. Rectal temperatures and blood samples were obtained at weaning and after relocation. Body weight was determined on d 1, at weaning, after relocation, and at weekly intervals for 5 wk thereafter. Blood chemistry profiles and serum cortisol concentrations were also determined. Rectal temperature and potassium increased and calcium decreased after groups I and II were transported. Glucose was lowest in group II. Total protein was greater in group I compared to group III. Albumin was greatest in group I compared to all other groups. Sodium was greatest in group II compared to all other groups. Anion gap was greatest in group II compared to group IV. Cortisol, phosphorus, gamma-glutamyltransferase, and chloride, were greater in groups I and II after transportation. In summary, transportation impacted several physiological indicators of health and well-being in weaned pigs, and providing SDPP prior to weaning prevented transportation-induced changes in sodium, glucose, and anion gap levels. / Master of Science

Les truies Yorkshire-Landrace doivent être saillies avant 260 jours d'âge afin de s'assurer qu'elles ne soient ni trop lourdes et/ou ni trop grasses au moment de la saillie, et ainsi obtenir une bonne productivité. La longévité des truies pour les quatre premières parités est influencée par l'âge et le poids à la première mise bas. Le poids des truies augmente de manière quadratique, alors que l'épaisseur de gras dorsal demeure constante d'une parité à l'autre, pour un même stade physiologique. Une perte d'appétit en lactation a pour effet d'augmenter de façon linéaire les pertes de poids ou d'épaisseur de gras dorsal durant cette période. D'ailleurs, plus la perte de poids durant la lactation est importante, plus l'intervalle sevrage saillie fécondante suivant cette lactation est élevée pour certaines parités. Cette étude a donc permis d'établir certaines recommandations permettant d'optimiser davantage la productivité et la longévité des truies Yorkshire-Landrace.

Circoviren sind kleine, unbehüllte Viren mit einem einzelsträngigen zirkulären DNA Genom mit eine Größe von 1,7 bis 2,4 kb. Das Porcine Circovirus Typ 2 (PCV2), welches zum Genus Circovirus gehört, ist mit einer Anzahl von Krankheitsmanifestationen verbunden worden, die heute als Porcine Circovirus Assoziierte Krankheiten (PCVAD) zusammengefasst sind. Die PCV2-Infektion bei Rindern ist bis zum jetzigen Zeitpunkt marginal erforscht worden. Serologische Untersuchungen auf Circovirus spezifische Antikörperführten zu widersprüchlichen Ergebnissen. Im Jahr 2007 wurde von der Bovinen Neonatalen Panzytopenie (BNP) in Europa mit unklarer Genese berichtet. Das klinisch - pathologische Bild der Hämorrhagien ähnelte dem Krankheitsbild der Infektiösen Anämie, welche durch ein Circovirus bei Hühnern verursacht wird. Deshalb wurde in dieser Studie eine Breitspektrum PCR zum Nachweis von Cirocvirus-Genomen durchgeführt. In 5 von 25 BNP betroffenen Kälbern konnte circovirale DNA nachgewiesen werden. Das komplette Genom wurde nachfolgend amplifiziert, kloniert und sequenziert. Das nachgewiesene Genom (PCV2-Ha08) hat eine Länge von 1768 Nukleotiden und zeigte eine hohe Homologie (bis zu 99%) mit PCV2-Genotyp b (siehe Publikation 1). Als Ursache der BNP ist vor kurzen die Übertragung von Alloantikörpern über das Kolostrum beschrieben wurden, welche die Zerstörungen von Leukozyten und Thrombozyten sowie deren Vorläuferzellen bewirken. Ungeachtet dessen war es wichtig, die Empfänglichkeit und Immunantwort von Kälbern nach experimenteller Infektion mit PCV2 zu studieren. Für diesen Zweck wurden weitere 181 Proben von BNP-Kälbern aus Deutschland mit Hilfe einer Breitspektrum-PCR getestet. In zwei von 181 Proben wurde PCV2 DNA nachgewiesen. Die vollständigen Sequenzen konnten amplifiziert werden. Während das erste Genom aus einer Blutprobe eines Kalbs in Bayern stammte (PCV2-Ha09), stammte das zweite nachgewiesene Genom aus Lunge und Gehirn von einem Kalb in Sachsen (PCV2-Ha10). Das Genom (PCV2-Ha09) besteht aus 1768 nt, währenddessen das Genom (PCV2-Ha10) aus 1767 nt aufgebaut ist (siehe Publikation 2). Weiterhin wurden die PCV2 Empfänglichkeit und die Immunantwort von Kälbern durch experimentellen PCV2 Inokulation sowie die Möglichkeit, eine Serokonversion nach Impfung mit einer kommerziellen PCV2 Vakzin zu entwickeln, untersucht. PCV2-spezifische Antikörper wurden in den PCV2-infizierten Tieren und in den PCV2-immunisierten Tieren im Tag 11 und 7 nach Inokulation (p.i.) nachgewiesen. PCV2-Genome wurden durch quantitative Realtime-PCR zwischen Tag 4 und Tag 46 p.i. nur in den Blutproben sowie in verschiedenen Geweben (z.B. Milz, Lymphknoten, Thymus) der PCV2-infizierten Tiere nachgewiesen. Das Genom, welches von den Lymphknoten der PCV2-infizierten Kälber erneut isoliert wurde, zeigt eine Identität von 99,9% gegenüber dem Inokulum. Dies weist möglicherweise auf adaptierte Mutationen im PCV2 Genom hin. Die Mutationen C1708T und G365C sind während der Infektionen aufgetreten. Die Sequenzanalyse zeigt eine mögliche adaptierte Mutation an der Aminosäure Nr. 105 in Replikationsgen (Met zu Ile) (siehe Publikation 3). Zusammenfassend kann geschlussfolgert werden, dass der Nachweis der PCV2 Genomen und eine experimentell induzierte Serokonversion möglich war. Es konnte gezeigt werden, dass die Empfänglichkeit von PCV2 nicht allein auf Schweine begrenzt ist und eine Übertragung von PCV2 auf Rinder möglich ist.

Made available in DSpace on 2015-03-26T13:47:03Z (GMT). No. of bitstreams: 1 texto completo.pdf: 914989 bytes, checksum: 6324bfc7a5398520ac5042a3539b706e (MD5) Previous issue date: 2011-11-22 / Porcine circovirus-2 (PCV2) has been related as the causative agent of the Postweaning Multissystemic Wasting Syndrome (PMWS) and other diseases called porcine circovirus associated diseases (PCVAD). They are responsible for economic losses in pork production worldwide. There is only a few scientific studies describing the infection in other species but swine and their hole at the epidemiologic dynamics of the diseases related to the PCV2. The aim of this study is to investigate the occurrence of infection by the PCV2 in wild mice (Mus musculus and Rattus rattus) captured in hog farms. The capture of the 40 sorted mice was made at 5 pig wean tofinish farms in Minas Gerais, an important state of pork production in Brazil. Samples of tissues (lymph nodes, spleen, kidney, heart and lungs) and blood were collected from the mice. The tissue fragments collected were submitted to immunohistochemistry and Nested PCR. Additionally, samples from spleen and lungs were analyzed by histology assays. Presence of antibodies anti-PCV2 was tested by ELISA assays. Immunohistochemical analysis showed positive prints in 12 animals, mostly on spleen (sub scapular area), lungs (alveolar macrophages) and kidney (inside the tubules). The 12 serum analyzed by ELISA hasn t detected antibodies anti-PCV2. Histopathological analyses revealed in some samples, a multifocal and lympho-neutrophilic interstitial bronchopneumonia, with some node formations. Moreover, spleen samples showed a mild to moderate lymphocyte depletion related to the PCVAD. The Nested PCR assays showed the presence of viral DNA at different tissues from 6 tested rodents. Thus, the results found in this work, indicate that mice from the species Mus musculus and Rattus rattus can be naturally infected by the PCV2 and they would play a hole in the epidemiology of PCVAD. However, more studies are necessary to confirm the transmission of the PCV2 from wild rodents to pigs. / O porcine circovirus-2 (PCV2) é atribuído como um dos agentes relacionados a doenças associadas ao circovírus (PCVAD), ocasionando perdas econômicas significativas na produção mundial de suínos. Poucos trabalhos são realizados a respeito da infecção em outras espécies pelo PCV2 e sua participação na epidemiologia das doenças associadas ao vírus. O propósito desse estudo foi investigar a ocorrência de infecção em roedores peridomésticos das espécies Mus musculus e Rattus rattus pelo PCV2 em granjas comerciais de suínos. Animais dessas espécies foram capturados em importantes centros de produção no estado de Minas Gerais. Amostras de órgãos (linfonodos, baço, rins, fígado, pulmão) e sangue foram coletadas. Os fragmentos de tecidos coletados foram submetidos ao teste de imunohistoquímica e Nested PCR. Adicionalmente, foram realizadas avaliações histológicas em amostras de baço, rim e pulmão. Presença de anticorpos anti-PCV2 foram avaliados pela técnica de ELISA. O teste de imunohistoquímica demonstrou marcações encontradas em 12 animais, principalmente no baço (região subcapsular), no pulmão (macrófagos alveolares) e nos rins (interior dos túbulos). A análise do soro pela técnica de ELISA não detectou anticorpos contra o PCV-2 nas 12 amostras avaliadas.. A histopatologia demonstrou em algumas amostras, uma pneumonia bronco-intersticial neutrofílica e linfocítica, multifocal e moderada, com formação de nódulos linfóides associados a vasos e bronquíolos. No ensaio de nested-PCR foi detectado DNA viral em diferentes tecidos avaliados de seis animais. Os resultados citados demonstram que os roedores domésticos das espécies estudadas podem exercer importante papel na epidemiologia das doenças relacionadas ao PCV2. No entanto, mais estudos são necessários para comprovar a transmissão do PCV2 dos roedores para os suínos.

Porcine circovirus associated disease (PCVAD) is an umbrella term for a multitude of diseases and syndromes that have a negative impact on the health and economics of pig production operations throughout the world. Porcine circovirus type 2 is the causative agent of PCVAD; however the presence of PCV2 alone is rarely enough to cause clinical disease. In order for the full development of PCVAD the presence of a co-infecting pathogen is required. The mechanisms by which co-infection leads to disease remain ongoing areas of research, but it is thought that host immune modulations by PCV2 or a co-infecting pathogen are critical in the pathogenesis of PCVAD. In the first study of this dissertation the ability of PCV2 to induce regulatory T-cells (Tregs) and alter cytokine production was evaluated in vivo. The addition of PCV2 to a multiple viral challenge resulted in a significant increase in Tregs. Levels of IL-10 and IFN-γ were also found to be altered when PCV2 was added to a multiple viral challenge. In further experiments, monocyte derived dendritic cells (MoDC) were infected with different combinations and strains of PCV2 and PRRSV in vitro and evaluated for expression levels of programmed death ligand-1 (PD-L1), IL-10, CD86, swine leukocyte antigen-1 (SLA-1), and swine leukocyte antigen-2 (SLA-2). Expression levels of PD-L1 were significantly increased in PCV2 and PRRSV co-infected MoDCs. SLA-1, SLA-2, and CD86 expression levels were significantly decreased in the MoDC treatment groups containing both PCV2 and virulent stains of PRRSV. MoDC IL-10 expression was significantly increased by PCV2 and virulent strains of PRRSV co-infection. Finally, we investigated the role of the PD-L1/programmed death ligand-1 (PD-1) axis in porcine lymphocyte anergy, apoptosis, and the induction of Tregs. Lymphocyte populations with normal PD-1 expression had significantly higher percentages of anergic and apoptotic lymphocytes, and CD4+CD25HighFoxP3+ Tregs when compared to a PD-1 deficient lymphocyte population. The findings from these studies indicate host immune modulation by PCV2 in vivo and the development of a regulatory phenotype of dendritic cell following PCV2/PRRSV co-infections in vitro that may contribute to a dysfunctional adaptive immune response and the overall pathogenesis of PCVAD. / Ph. D.

In der Schweinezucht werden konventionell 1-3 x109 Spermien als Frischsamen in einem Volumen von 80-100 ml intrauterin versamt. Im Vergleich zur Rinderbesamung, bei der mitunter 2 x106 Spermien zu Trächtigkeiten führen, sind Ejakulate von Ebern ineffizient. Die einzige Möglichkeit geringe Spermienzahlen erfolgreich zu versamen besteht darin die Spermienablage nahe dem Ort der Befruchtung im distalen Isthmus des Eileiters durchzuführen. Die spezies-spezifische Bindung von Säugerspermien an diverse Oberflächenzellen des weiblichen Traktes, die der Kapazitation und Hyperaktivierung vorangehen, setzt die Erkennung von Kohlenhydraten durch lektin-ähnliche Proteine der Spermienplasmamembran voraus Daher wurde vermutet, dass eine mutmaßliche Bindung porziner Spermien an das Uterusepithel ebenfalls Protein-Kohlenhydrat vermittelt ist. Ziel der vorliegenden Arbeit war die Etablierung eines Zellkulturmodells aus primären Uterusepithelien der Sau (Sus scrofa), um mögliche Gründe für die hohen Spermienzahlen in der Schweinbesamung zu untersuchen und zu identifizieren. Porzine uterine Epithelien wurden mittels Verdau mit Trypsin/EDTA (1x) gewonnen. Zellen wurden auf Collagen (rat tail, type I) auf Kollagen-beschichteten Deckgläschen aus Glas in 6-Well Kulturschalen ausgesät. Die Anheftung der Zellen an die Kollagen-Matrix wurde nach 12-36 Stunden beobachtet. Erste Kolonien bildeten sich nach fünf bis sieben Tagen und Konfluenz wurde nach zehn bis fünfzehn Tagen erreicht. Der Nachweis der Zellart erfolgte mittels Immunfluoreszenz-Färbung mit einem Epithelien-spezifischen Antikörper (anti-Cytokeratin-19). Eberspermien banden innerhalb weniger Minuten an die UEC und verblieben währenddessen motil. Eine verringerte Spermienbindung wurde sowohl auf Aortenendothelien als auch fötalen Fibroblasten des Schweins beobachtet. Dies weist auf das Vorhandensein spermienspezifischer Liganden auf den UEC hin. Spermien, wie auch UEC wiesen hohe Bindungsintensitäten für Lektine die an Glc-NAc, (WGA) sowie Gal-NAc (sWGA) binden. Die Sättigung von Sialinsäure führte zu einer stark verminderten Spermienbindung auf den UEC. Bei einer Sättigung von Glc-NAC wurde kein Unterschied beobachtet. Dies kennzeichnet Sialinsäure als den vermutlich wichtigsten Kohlenhydratrest in der Spermien-Epithelzellbindung.