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.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233 amino acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs, and compare these responses to pigs diagnosed with porcine circovirus-associated disease (PCVAD). The approach was to react porcine sera with different CP polypeptide fragments that each contained one or more immunoreactive regions. Expression of polypeptides was performed using E.coli. Initial results showed that sera from vaccinated pigs preferentially recognized only the largest CP(43-233) polypeptide fragment and showed low levels of binding to other CP polypeptide fragments. The results of sera from pigs diagnosed with PMWS showed only minimal reactivity with CP polypeptide fragments, including the largest CP(43-233). PCV2 infected or PDNS diagnosed pigs reacted to all CP polypeptides: however, the strongest reactivity was primarily directed towards CP polypeptides containing residues in the 160-180 region. For this purpose, finer mapping studies were performed. These experiments involved reacting sera from experimentally infected PCV2 pigs and PDNS pigs with overlapping oligopeptides that covered amino acids 141-200. Overall, the results showed a subset of experimentally infected pigs and pigs with PDNS preferentially recognized the CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175 and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The results from this study support the notion of PCV2 modulation of immunity. Furthermore, the methods incorporated in this study provide a means for characterizing the immune response upon vaccination, natural infection and disease.

O Circovírus suíno tipo 2 (PCV-2) pertence ao gênero Circovirus da família Circoviridae. É considerado “vírus emergente", tendo sido associado, principalmente, à Síndrome de Refugagem Multissistêmica dos suínos (SRM). O genoma circular é composto por: (i) ORF-1 que codifica a proteína replicativa; (ii) ORF-2 que codifica a proteína estrutural formadora do capsídeo, (iii) região não codificadora que intercala as ORF-1 e 2 e contem a origem da replicação, denominada IGS-1 e (iv) região que intercala as ORF-1 e 2, denominada IGS-2. Oito amostras, denominadas amostras completas, tiveram mais de 1705 nucleotídeos seqüenciados (945 da ORF-1, 699 da ORF-2, 20 da IGS-1 e 39 da IGS-2); duas amostras tiveram em média 1692 nucleotídeos seqüenciados (945 da ORF-1 e 699 da ORF-2, os restantes correspondem às regiões IGS-1 e 2); uma amostra teve 1392 nucleotídeos seqüenciados (945 da ORF-1, 414 da ORF-2, 9 da IGS-1 e 24 da IGS-2) e nove amostras tiveram em média 970 nucleotídeos seqüenciados (196 da ORF-1 e 699 da ORF-2, os restantes correspondem às regiões IGS-1 e 2). Portanto, a partir dessas 20 amostras em estudo, foram obtidas: (i) oito amostras completas; (ii) 11 seqüências completas de ORF-1 e (iii) 19 seqüências completas de ORF-2, as quais foram analisadas. A identidade de nucleotídeo variou de: (i) 99,7% a 100% entre as oito amostras completas; (ii) 99,3% a 100% entre as 11 seqüências completas de ORF-1 e (iii) 91,9% a 100% entre as 19 seqüências completas de ORF-2. A topologia das árvores genealógicas utilizando as oito seqüências completas e as 11 seqüências completas de ORF-1 foi similar, agrupando todas as amostras em estudo em um só grupo denominado subtipo PCV-2a. Pela análise da genealogia da ORF-1 observou-se que todas as amostras agruparam-se com uma amostra de PCV-2 associada à Síndrome de Dermatite e Nefropatia suína (PDNS), formando um grupo separado das amostras de PCV-2 associadas à Síndrome de Refugagem Multissistêmicas dos suínos (SRM) e abortamento. A genealogia proposta para as 19 amostras que tiveram a ORF-2 seqüenciada, dividiu as amostras em dois grupos, sendo que 14 amostras agruparam-se num grupo denominado subtipo PCV-2a e 5 no subtipo PCV-2b. Os resultados mostraram a circulação de, pelo menos, dois subtipos de PCV-2 no Brasil / Porcine circovirus 2 belongs to Circovirus genus and Circoviridae family. It is considered an “emerging virus" being associated to Postweaning Multisystemic Wasting Syndrome (PMWS). The circular viral genome is formed by: (i) ORF-1 coding for the replicative associated proteins; (ii) ORF-2 encoding the structural proteins of the viral capsid; (iii) a non-coding intergenic sequence between ORF-1 and ORF-2 containing the replicative origin of the viral genome (IGS-1) and (iv) a second non-coding intergenic sequence between ORF-1 and ORF-2 (IGS-2). Eight samples, named complete sequences, had about 1705 nucleotides determined (945 from ORF-1, 699 from ORF-2, 20 from IGS-1 and 39 from IGS-2); two samples had about 1692 nucleotides sequenced (945 from ORF-1, 699 from ORF-2, and the rest from IGS-1 and 2); one sample had 1392 nucleotides sequenced (945 from ORF-1, 414 from ORF-2, 9 from IGS-1 and 24 from IGS-2) and nine samples had about 970 nucleotides sequenced (196 from ORF-1, 699 from ORF-2, and the rest from IGS-1 and 2). Therefore, from the 20 samples it was obtained: (i) eight complete sequences; (ii) 11 complete sequences of ORF-1 and (iii) 19 complete sequences of ORF-2. The identity at nucleotide level was from: (i) 99,7% to 100% among the eight complete sequences; (ii) 99,3% to 100% among the 11 sequences of ORF-1 and (iii) 91,9 to 100% among the 19 sequences of ORF-2. The topology of the tree generated by the ORF-1 analysis revealed a cluster formed by the Brazilian samples of PCV-2 and the samples associated to PDNS and another cluster formed by the PCV-2 associated to other syndromes. The genealogy presented for the 19 samples using the data from ORF-2 revealed the presence of two clusters, one of them formed by 14 samples named subtype PCV-2a and the other formed by 5 samples, named PCV-2b. The results demonstrated that, at least, two subtypes of PCV-2 circulate in Brazil

O Circovírus suíno tipo 2 (PCV-2) pertence ao gênero Circovirus da família Circoviridae. É considerado “vírus emergente”, tendo sido associado, principalmente, à Síndrome de Refugagem Multissistêmica dos suínos (SRM). O genoma circular é composto por: (i) ORF-1 que codifica a proteína replicativa; (ii) ORF-2 que codifica a proteína estrutural formadora do capsídeo, (iii) região não codificadora que intercala as ORF-1 e 2 e contem a origem da replicação, denominada IGS-1 e (iv) região que intercala as ORF-1 e 2, denominada IGS-2. Oito amostras, denominadas amostras completas, tiveram mais de 1705 nucleotídeos seqüenciados (945 da ORF-1, 699 da ORF-2, 20 da IGS-1 e 39 da IGS-2); duas amostras tiveram em média 1692 nucleotídeos seqüenciados (945 da ORF-1 e 699 da ORF-2, os restantes correspondem às regiões IGS-1 e 2); uma amostra teve 1392 nucleotídeos seqüenciados (945 da ORF-1, 414 da ORF-2, 9 da IGS-1 e 24 da IGS-2) e nove amostras tiveram em média 970 nucleotídeos seqüenciados (196 da ORF-1 e 699 da ORF-2, os restantes correspondem às regiões IGS-1 e 2). Portanto, a partir dessas 20 amostras em estudo, foram obtidas: (i) oito amostras completas; (ii) 11 seqüências completas de ORF-1 e (iii) 19 seqüências completas de ORF-2, as quais foram analisadas. A identidade de nucleotídeo variou de: (i) 99,7% a 100% entre as oito amostras completas; (ii) 99,3% a 100% entre as 11 seqüências completas de ORF-1 e (iii) 91,9% a 100% entre as 19 seqüências completas de ORF-2. A topologia das árvores genealógicas utilizando as oito seqüências completas e as 11 seqüências completas de ORF-1 foi similar, agrupando todas as amostras em estudo em um só grupo denominado subtipo PCV-2a. Pela análise da genealogia da ORF-1 observou-se que todas as amostras agruparam-se com uma amostra de PCV-2 associada à Síndrome de Dermatite e Nefropatia suína (PDNS), formando um grupo separado das amostras de PCV-2 associadas à Síndrome de Refugagem Multissistêmicas dos suínos (SRM) e abortamento. A genealogia proposta para as 19 amostras que tiveram a ORF-2 seqüenciada, dividiu as amostras em dois grupos, sendo que 14 amostras agruparam-se num grupo denominado subtipo PCV-2a e 5 no subtipo PCV-2b. Os resultados mostraram a circulação de, pelo menos, dois subtipos de PCV-2 no Brasil / Porcine circovirus 2 belongs to Circovirus genus and Circoviridae family. It is considered an “emerging virus” being associated to Postweaning Multisystemic Wasting Syndrome (PMWS). The circular viral genome is formed by: (i) ORF-1 coding for the replicative associated proteins; (ii) ORF-2 encoding the structural proteins of the viral capsid; (iii) a non-coding intergenic sequence between ORF-1 and ORF-2 containing the replicative origin of the viral genome (IGS-1) and (iv) a second non-coding intergenic sequence between ORF-1 and ORF-2 (IGS-2). Eight samples, named complete sequences, had about 1705 nucleotides determined (945 from ORF-1, 699 from ORF-2, 20 from IGS-1 and 39 from IGS-2); two samples had about 1692 nucleotides sequenced (945 from ORF-1, 699 from ORF-2, and the rest from IGS-1 and 2); one sample had 1392 nucleotides sequenced (945 from ORF-1, 414 from ORF-2, 9 from IGS-1 and 24 from IGS-2) and nine samples had about 970 nucleotides sequenced (196 from ORF-1, 699 from ORF-2, and the rest from IGS-1 and 2). Therefore, from the 20 samples it was obtained: (i) eight complete sequences; (ii) 11 complete sequences of ORF-1 and (iii) 19 complete sequences of ORF-2. The identity at nucleotide level was from: (i) 99,7% to 100% among the eight complete sequences; (ii) 99,3% to 100% among the 11 sequences of ORF-1 and (iii) 91,9 to 100% among the 19 sequences of ORF-2. The topology of the tree generated by the ORF-1 analysis revealed a cluster formed by the Brazilian samples of PCV-2 and the samples associated to PDNS and another cluster formed by the PCV-2 associated to other syndromes. The genealogy presented for the 19 samples using the data from ORF-2 revealed the presence of two clusters, one of them formed by 14 samples named subtype PCV-2a and the other formed by 5 samples, named PCV-2b. The results demonstrated that, at least, two subtypes of PCV-2 circulate in Brazil

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.

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.

Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases facing the global swine industry. Porcine circovirus type 2 (PCV2) is the primary and essential causative agent of PCVAD, but development of clinical disease typically requires co-infection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV). The specific mechanisms of co-infection that lead to clinical disease are not fully understood, but immune modulation by the co-infecting viruses is thought to play a critical role. The ability of dendritic cells (DC) infected with PRRSV, PCV2 or both to induce regulatory T cells (Tregs) was evaluated in vitro. DCs infected with PCV2 significantly increased CD4+CD25+FoxP3+ Tregs (p<0.05) and DCs co-infected with PRRSV and PCV2 induced significantly higher numbers of Tregs than with PCV2 alone (p<0.05). This Treg induction was found to be dependent on TGF-β and not IL-10. Further investigation of the in vivo swine immune response to acute co-infection with PCV2 and PRRSV failed to detect activation of Tregs in peripheral blood mononuclear cells (PBMCs) or bronchoalveolar lavage samples. The Treg response to in vitro and in vivo PRRSV challenge in pigs persistently infected with PCV2 or vaccinated against PCV2 was evaluated. There was no significant difference in Tregs in PBMCs among chronically PCV2-infected, vaccinated PCV2 challenged or negative control pigs. However, following in vitro infection of monocyte-derived dendritic cells with PCV2, PRRSV, or both viruses, co-cultured lymphocytes from chronically infected and PCV2 vaccinated pigs had significantly (p<0.05) decreased Treg expression in the virus infected groups compared to the negative controls. In separate experiments, pigs vaccinated against PCV2 and subsequently challenged with an attenuated PRRSV strain and its pathogenic parental strain developed increased CD4+CD25+FoxP3+ Tregs (p<0.05) in PBMC samples compared to uninfected controls, and this correlated with increased suppressor activity and IL-10 expression. The findings from these studies indicate that the interaction of PCV2 and PRRSV in swine modulates the host immune response mediated in part through the activity of Tregs. However, the extent to which Tregs orchestrate a dysregulated immune response in the pathogenesis of PCVAD in vivo remains to be determined. / Ph. D.

The non-pathogenic porcine circovirus type 1 (PCV1) was originally isolated as a persistent contaminant of the porcine kidney cell line PK-15. Whereas, porcine circovirus type 2 (PCV2) causes postweaning multisystemic wasting syndrome (PMWS) in pigs, which is devastating to the swine industry. My objectives were to determine the effect of maternally derived antibodies on PCV2 infection, assess the role of 2 amino acid substitutions in the PCV2 capsid protein in PCV2 attenuation, evaluate the effect of Rep gene exchange between PCV1 and PCV2 on growth characteristics of a chimeric PCV2, and evaluate the role of open reading frame (ORF) 3 of PCV2 in virus replication and pathogenesis in pigs. Under field conditions, PCV2 infection is widespread and most breeding pigs are seropositive. Assessment of the role of PCV2 maternal antibodies in preventing PCV2 infection in piglets provided evidence that higher levels of maternal antibody provide more protection to piglets. Two amino acid substitutions in the PCV2 capsid protein that enhanced virus replication in vitro and attenuated the virus in vivo were evaluated for their pathogenicity in pigs. The results indicated that P110A and R191S are collectively responsible for virus attenuation. PCV1 replicates better in PK-15 cells and grows at least 1-log titer higher than PCV2. A chimeric PCV with the rep gene of PCV1 replacing that of PCV2 in the genomic backbone of PCV2 replicated more rapidly than PCV1 and PCV2, and more efficiently than PCV2, although to a titer similar to PCV1. The ORF3 of PCV2 is believed to encode a protein involved in apoptosis. The ORF3 start codon was mutated from ATG to GTG and the resulting mutant muPCV2 was infectious in vitro and in pigs; therefore ORF3 is dispensable for virus replication. The pathogenicity of muPCV2 was compared with PCV2 in vivo. Delayed viremia and seroconversion, decreased viral loads, lower level of IgG antibodies, and lower amounts of PCV2 antigen in mesenteric lymph nodes suggested attenuation of muPCV2. However, there was no significant difference in histological or gross lesions in tissues between PCV2- and muPCV2-inoculated groups. The role of ORF3 in attenuation needs to be further elucidated. / Ph. D.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal